Systems and methods for detecting animal pregnancy

ABSTRACT

Testing systems and methods are disclosed for detecting a pregnancy marker of an animal. A test kit may include a first standard with a first concentration of the marker, a second standard with a second concentration of the marker lower than the first concentration, and at least three test surfaces coated with a biomolecular recognition element selected to bind with the marker. The test may also include a reagent solution with a conjugated biomolecular recognition element that binds with the marker, and a visual indicator that produces a visually detectable change when reacting with the conjugated biomolecular recognition element bound to each test surface. A detectable change generated by the marker from the sample with an intensity greater than the first concentration yields a pregnant result, lower than the second concentration yields a not pregnant result, and between the first and second concentrations yields a retest result.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application to U.S. patent applicationSer. No. 13/038,248, filed Mar. 1, 2011, which is hereby incorporated byreference.

BACKGROUND

The present disclosure relates generally to test systems and methods fordetecting the presence of markers in animals. More specifically, thetest systems and methods of the present disclosure may be used to detecta pregnancy marker that indicates if an animal, such as a ruminant, ispregnant.

Traditional testing for determining the pregnancy of some animals, suchas ruminants, involves physical inspection of such an animal by aveterinarian. Traditional testing methods include using ultrasound andby rectal palpitation. However, these tests require an expert to performthe test, are time intensive, and are not cost effective.

Blood-based or other body fluid-based (i.e., milk) tests for detectingpregnancy markers provide another tool for aiding in early pregnancydiagnosis. In such tests, a sample of animal fluid can be withdrawnonsite, and then sent to an offsite laboratory for detection of a markerwhose presence indicates that an animal is pregnant. For example,BioTracking LLC offers a laboratory test service for detecting thepresence of a protein marker called pregnancy-specific protein B (PSPB)in ruminant animals. See U.S. Pat. Nos. 4,554,256 and 4,705,748, both ofwhich are incorporated herein by reference. This test requires sending ablood sample taken onsite from a ruminant, such as a cow, to alaboratory, where a sandwich ELISA test is used to detect the presenceof PSPB in the sample using quantitative means. PSPB includes severalmolecular weight and isoelectric variants of proteins. (Sasser andRuder, 1987 & Sasser et al., 1989). Therefore, a polyclonal antiserumcan be used to detect several of these protein variants of PSPB (Sasseret al., 1986). Additionally, PSPB molecules, referred to as pregnancyassociated glycoproteins (PAGs), have been found in several eutherianmammals (Placentalia); most specifically they have been found inArtiodactyla, Perissodactyla, Carnivora and Rodentia (Szafranska et al.,2006).

There is not currently available a simple, non-laboratory (i.e.,“on-the-farm”) test for detecting markers, such as a pregnancy marker,that a non-professional can fully administer in the field. A need existsfor a test that employs qualitative or semi-qualitative metrics fordetecting markers, including for detecting pregnancy markers todetermine if an animal is pregnant. A non-professional could use such atest in the field instead of requiring a professional laboratoryanalysis. An onsite test may also improve temporal efficiency bydecreasing the time from sample collection to result, potentially fromseveral days to hours or minutes.

Moreover, testing methods designed only to render a binary determination(e.g., positive/negative; reactive/non-reactive; and/orpregnant/not-pregnant) are limited in that they produce ambiguousresults under certain conditions. For example, when a pregnancy isaborted or terminates prematurely, certain pregnancy markers may stillbe present in the blood. Depending on the timing and method of detectionused, these markers may be detected creating a false positive result(e.g., a not pregnant animal being categorized as pregnant). Falsepositives are detrimental to reproductive management decisions astemporal efficiency is decreased (e.g., takes longer to correctlyidentify the pregnancy status of the animal).

When using a binary approach, a main way to improve and optimize testresults is by adjusting the sensitivity and specificity of the test. Forpregnancy testing, sensitivity may be the percentage of pregnant animalscorrectly identified as pregnant. Specificity may be the number of notpregnant animals correctly identified as not pregnant. The problem isthe sensitivity and specificity for a binary classification is connectedsuch that an improvement in one parameter can be offset by decreasedperformance in the other. For example, a cutoff that results in moreefficient identification of not pregnant animals (improved specificity)can be offset by having more unintentional hormone induced abortions dueto more false negatives (lower sensitivity). A cutoff resulting inbetter identification of pregnant animals (improved sensitivity) andless induced abortions can be offset by decreased efficiency for takinglonger to identify a portion of not pregnant animals due to more falsepositives (lower specificity).

Under the binary approach, there is therefore some probability of falsepositives and false negatives no matter what signal is set as the binarytest standard. Setting the test standard at a cutoff yielding 100%sensitivity will increase the probability of false positives. Settingthe test standard cutoff to yield 100% specificity will increase theprobability of false negatives. Setting a cutoff between 100%sensitivity and 100% specificity may allow for some optimization, butthere will remain some probability of false positive and false negativeresults.

A need exists for a test that can minimize the issues associated withspecificity and sensitivity for binary tests. A need exists for apregnancy test for early identification of non-pregnant animals withlimited misidentification of pregnant animals. The sooner a decision canbe made following previous pregnancy and subsequent breeding, the morebenefit is attained.

SUMMARY

The present disclosure may include systems and methods for detecting oneor more markers. The marker being detected may be a pregnancy markerpresent in an animal when the animal is pregnant. The method may includeselecting a marker, preparing a test surface for detecting the marker,preparing first and second test standards that may respectivelycorrespond to a first amount of marker and second amount of markerdifferent from the first amount of marker, obtaining a sample from theanimal, testing the sample on the test surface, comparing the magnitudeof a signal corresponding to the level of marker in the tested sampleagainst the first and second standards, and categorizing the animalbased on the tested sample, for example, as positive/reactive (e.g.,pregnant), negative/non-reactive (e.g., not pregnant), or recheck.Recheck may indicate that another sample should be retrieved at a timeafter the initial sample was retrieved to determine a reading, forexample, of positive/reactive (e.g., pregnant), negative/non-reactive(e.g., not pregnant), recheck again, etc.

A kit of the present disclosure may include one or more test surfacescoated with a biomolecular recognition element (e.g. a detectionantibody or antigen, etc.) specific to the marker of the animal beingdetected, a first standard or control that may provide an indicationconsistent with a first amount of the detected marker found in a sample,a second test standard that may provide an indication consistent with asecond amount of marker found in a sample, and one or more solutionswith reagents for performing the test. The test kit may be provided to atester for performing the test (e.g. pregnancy test) in the field todetect the marker in a sample of an animal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart illustrating a method of detecting and analyzingthe presence of a marker in an animal according to the presentdisclosure.

FIG. 2 illustrates embodiments of testing kits of the present disclosurefor detecting a marker that may each include one or more testingsurfaces, one or more standards, and one or more reagents.

FIGS. 3 (a-b) are a flow chart illustrating embodiments of methods ofdetecting and analyzing the presence of a marker in an animal accordingto the present disclosure.

FIG. 4 shows an embodiment of an assay with test surfaces including afirst standard of a marker, a second standard of the marker, and testsamples from one or more animals to compare qualitatively against thefirst and second standards.

DETAILED DESCRIPTION

As shown in FIG. 1, the present disclosure may include a testing method10 for detecting one or more markers. The marker being detected may be apregnancy marker present in an animal when the animal is pregnant. Themethod may include selecting a marker 12, preparing a test surface fordetecting the marker 14, preparing first and second test standards thatmay respectively correspond to a first amount of marker and secondamount of marker different from the first amount of marker 16, obtaininga sample from the animal 18, testing the sample on and/or with the testsurface 20, comparing the magnitude of a signal corresponding to thelevel of marker in the tested sample against the first and secondstandards 22, and categorizing the animal based on the tested sample,for example, as positive/reactive (e.g., pregnant),negative/non-reactive (e.g., not pregnant), or recheck. Recheck mayindicate that another sample should be retrieved at a time after theinitial sample was retrieved to determine a reading, for example, ofpositive/reactive (e.g., pregnant), negative/negative (e.g., notpregnant), recheck again, etc.

As shown in FIG. 2, the present disclosure may include a test kit, suchas Kit A, Kit B, Kit C, and/or Kit D, for detecting one or more markers.The kit may include one or more test surfaces coated with a biomolecularrecognition element (e.g., a detection antibody, antigen, etc.) specificto the marker of the animal being detected, a first standard or controlS₁ that may provide an indication consistent with a first amount of thedetected marker found in a sample, a second test standard S₂ that mayprovide an indication consistent with a second amount of marker found ina sample, and one or more solutions R (e.g., R_(1a), R₂,R₃ for Kit A)with reagents for performing the test. The test kit may be provided to atester for performing the test (e.g. pregnancy test) in the field todetect the marker in a sample of an animal.

The methods and systems (e.g., kits) of the present disclosure will bedescribed in detail below. While the detection of pregnancy markers inanimals is used as an illustrative example throughout, it will beappreciated that the systems and methods of the present disclosure canalso be applied to detecting markers for other types of testing ordiagnostic procedures, including testing animals for the presence ofmarkers associated with caprine arthritic encephalitis (CAE), bovineviral diarrhea (BVD), etc.

Selection of Marker(s) to Detect

Using the methods and systems of the present disclosure, several typesof markers can be detected. The marker being detected may indicatewhether an animal is pregnant or has a particular disease or condition.The marker being detected may be a protein, a lipid, a carbohydrate,etc. The marker being detected may depend on the type of animal beingtested, the disease, condition, etc. being tested, the format of thetest (i.e., assay, strip, etc.), and/or the type of biomolecularrecognition element (e.g. antibodies, antigens, etc.) being used todetect the marker. The marker being detected may be a single marker(e.g., a protein marker) or a combination of markers (e.g., a proteinmarker and a steroid marker; two different protein markers; two variantsof the same protein marker, etc.). The marker being detected may bespecific to one condition (e.g., pregnancy) or multiple conditions(e.g., pregnancy and a disease)

In some embodiments, the marker being detected, when present inincreased or increasing amounts, may indicate a positive/reactive (e.g.,pregnant) result. In some embodiments, the marker being detected, whenabsent or present in decreased or decreasing amounts, may indicate apositive/reactive (e.g., pregnant) result.

As an example, there are several pregnancy markers that may be detectedto indicate if an animal is pregnant.

PROTEIN MARKERS: In some embodiments, a protein marker may be detectedfor determining pregnancy. The protein marker may be a protein from theaspartic acid protease family. The protein marker may be pregnancyspecific protein B (PSPB) or any of the PSPB variants (see Sasser et al.(1987) that identifies about 42 variants), and/or any of thepregnancy-associated glycoprotein (PAG) molecules. Several variants ofPAG are known, including PAG-1, PAG-2, PAG-3, PAG-4, PAG-5, PAG-6,PAG-7, PAG-9, PAG-16, PAG-18, PAG-19, PAG-55, and MON PAG (see U.S. Pat.Nos. 6,869,770; 7,393,696; and 7,575,861, all of which are incorporatedby reference).

It is believed that all of the PAG variants may be molecular forms ofPSPB. PSPB is a protein isolate (a mixture of proteins). PAG was termedat a later time than was PSPB, and some, many, or all of the individualPAG's may be present in the PSPB protein fraction (see Butler et al.1982; Zoli et al. 1991; and Hughes et al. 2003).

PSPB and PAG's belong to a large family of aspartic peptidases expressedin the placenta of species in the Artiodactyla order (e.g., cow, sheep,goat, pig, bison, deer, elk, moose, bighorn sheep, mountain goat, cameland other wild ungulates (split hoofed)) (see Hughes et al., 2003). Theyare also found in the Perissodactyla order (e.g., horse) and Carnivoraorder (e.g., cat). Hence, the systems and methods of the presentdisclosure that employ PSPB and/or PAG can be used to detect thepregnancy of any of these animals, or any other animal in which PSPBand/or PAG is found.

In some embodiments, measuring the concentration of the protein markerfound in a sample (e.g., blood, serum, plasma, etc.) retrieved from ananimal may indicate whether the animal has a level of the protein markerin the sample consistent with being pregnant. For example, aconcentration of PSPB that is less than or equal to between about 0.01nanograms per milliliter (ng/ml) to about 1.0 ng/ml of PSPB in serum ofan animal may indicate the animal is not pregnant. A concentration ofPSPB that is more than or equal to between about 0.1 ng/ml to about 1.5ng/ml of PSPB in serum of an animal may indicate the animal is pregnant.

To obtain an accurate pregnancy reading based on the selected marker,the sample may need to be retrieved from the animal at a specific timeafter conception or insemination. For example, on or after about 30 daysafter conception/insemination, a sample concentration equal to or belowabout 0.2 ng/ml of PSPB in ruminant serum may indicate the ruminant isnot pregnant, and equal to or above about 0.4 ng/ml of PSPB in ruminantserum may indicate that the ruminant is pregnant. A level between about0.2 ng/ml and about 0.4 ng/ml may indicate that the animal should berechecked at a later time to determine if the animal is pregnant.

In some embodiments, depending on what form of PSPB/PAG is beingdetected, and what type of pregnancy marker detector (e.g., antibody) isbeing used for the detection, the sample may be withdrawn earlier than30 days post-insemination. For example, some variants of PSPB/PAG have adifferent expression profile in the blood than other variants ofPSPB/PAG, which may allow for accurate detection of pregnancy earlierthan 30 days (see Sasser et al. (1986)).

Other protein markers that can be detected to determine pregnancyaccording to the present disclosure include bovine antigen glycoprotein(BAG); early pregnancy factor (EPF); early conception factor (ECF);pregnancy-specific glycoprotein (PSG60); pregnancy serum protein 60(PSP-60); and/or any of the interferon stimulated gene proteins (ISGPs)and/or the interferon-tau induced proteins, which include myxovirusresistance genes (e.g. MX1), UCRP, ISG-15, ISG-17, GCP-2,2′,5′oligoadenylate synthetase 1 (OAS 1), beta 2-microglobulin, IRF-1,IRF-2, I-8U, 1-8D Leu-13/9-27, and COX-2 (see U.S. Patent App. No.2008/0026384, which is incorporated herein by reference).

For example, MX1, MX2, Oas 1, and ISG-15 are interferon stimulated geneproteins that may be used as markers for detecting pregnancy. Theproteins are produced in response to the presence of interferon tau,which is a product of the trophoblast. The presence of interferon taumay provide a signal to the maternal system that an embryo is in theuterus. The messenger RNA for MX and ISG15 may arise at about 15 daysafter conception and remain in cattle until about 30 days to about 40days after conception. Ovine ISG15 mRNA may be detectable on about day13 of pregnancy, peak around about day 15, and remain high through aboutday 19. The effect of interferon tau stimulation is to continue thepresence of the MX or ISG-15 messenger RNA until about 35 days afterconception. The interferon tau response described above is for ruminantanimals (sheep, cows, goats, etc.). It is suggested that MX and ISG15protein may be used to detect pregnancy in cows (and other ruminantssuch as sheep) and gilts (pigs). (Hicks et. al. 2003).

STEROID MARKERS: In some embodiments, a steroid marker such asprogesterone or estrone sulfate may be detected for determiningpregnancy. Progesterone is present during the pregnancy of all eutherian(placental) mammals and is required to maintain pregnancy. Progesteroneis present during the reproductive cycle but is absent when the animalis in heat. If conception occurs, progesterone may remain high and notdecline as it would at the end of a reproductive cycle and during heat.Accordingly, the amount of progesterone in the blood of an animal at thetime of an expected next heat may be high if pregnant and low if notpregnant. The absence of progesterone in the blood may confirmnon-pregnancy and/or that an animal is in heat.

For example, in some types of tests for cattle, a level at or belowabout 1 ng/ml to about 2 ng/ml of progesterone in serum may indicatethat the animal is in, or near, heat and is not pregnant. A level at orabove about 2 ng/ml to about 6 ng/ml of progesterone in serum mayindicate the animal is pregnant. A level between about 1-2 ng/ml toabout 2-4 ng/ml may indicate that the animal should be rechecked at alater time to determine if the animal is pregnant. Progesterone levelscan be measured to determine the pregnancy of cattle about 18 to 24days, and perhaps preferably 20 to 22 days, afterconception/insemination.

In other embodiments, an estrogenic hormone such as estrone sulfate maybe detected for determining pregnancy. Estrone sulfate is a pregnancymarker for several mammals that is produced by the fetal/placental unitand can be measured in, for example, a blood sample retrieved from themother. This hormone is produced by the fetal/placental unit and doesnot arise until the late first to early second trimester of gestation.The level of estrone sulfate can be measured in cattle and horses, andin other animals, such as pigs, goats, sheep, alpaca, llama, etc., todetermine pregnancy.

For example, in cows, the level of estrone sulfate increases during theperiod of gestation. On or after about 100 days of gestation, the levelof estrone sulfate present in a cow may be measured to determinepregnancy. Accordingly, estrone sulfate levels can be measured todetermine the pregnancy of cattle about 100 days afterconception/insemination.

For horses, levels of estrone sulfate are different at different timesof gestation. On or after about 70 days of gestation, the level ofestrone sulfate in a horse can be measured to determine pregnancy.Accordingly, estrone sulfate levels can be measured to determine thepregnancy of horses about 70 days after conception/insemination.

In some embodiments, on or after about 35 days afterconception/insemination, a level at or below about 5.0 ng/mL of estronesulfate in serum may indicate the horse is not pregnant. On or afterabout 70 days after conception/insemination, a level at or below about9.4 ng/ml of estrone sulfate in serum may indicate the horse is notpregnant. On or after about 70 days after conception/insemination, alevel at or greater than about 13 ng/ml of estrone sulfate in serum mayindicate the horse is pregnant. On or after about 70 days afterconception/insemination, a level between about 9.5 ng/ml and about 13ng/ml of estrone sulfate in serum may indicate that another sampleshould be retrieved from the horse at a later time after the initialsample was taken to determine pregnancy.

Selection of Biomolecular Recognition Elements Specific to Marker(s)Being Detected

An appropriate biomolecular recognition element (BRE) may be selectedfor use depending on the marker(s) being detected. Any BRE that iscapable of allowing detection of a selected marker can be used. Forexample, the marker may be a molecule, receptor, antibody, antigen,nucleic acids, etc. The BRE may detect a combination of differentmarkers (e.g., a protein marker and a carbohydrate marker, more than oneprotein marker, more than one variant of a marker, etc). BRE(s) specificto multiple markers of one disease or condition, or to differentdiseases or conditions, may be used in combination if desired.

In some embodiments, a preferred BRE may be a detection antibody. Thedetection antibody may be polyclonal or monoclonal. Accordingly,detection antibodies are used as an illustrative, example below fordetecting markers. It will be appreciated that detection antibodies canbe substituted in the discussion below with any other appropriate BREcapable of allowing detection of the marker being detected.

For example, when detecting the pregnancy marker of PSPB, the BRE may bea detection antibody selected that is specific to PSPB. The detectionantibody may be, or include, polyclonal rabbit immunoglobulin (IgG),polyclonal goat IgG, polyclonal sheep IgG, polyclonal mouse IgG,monoclonal mouse or rabbit IgG, etc. In some embodiments, the detectionantibody may detect either a single variant or multiple variants ofPSPB/PAG.

Preparation of Test Surfaces for Conducting Test

There may be provided a test or support surface used for performing atest for detecting the presence of a selected marker(s). The test orsupport surface may be coated with/hold the selected detectionantibodies, etc. specific to the marker(s) being detected.

The support surface may be any surface on which the selected detectionantibodies, etc. can be coated/held for detection of the selectedmarker(s). In some embodiments, the test or support surface may be partof an assay having one or more containers (or wells). The test orsupport surface may be the inner surface of a well or container. Theinner surface of one or more wells or containers may be coated with thedetection antibody specific to the marker(s) being detected. Forexample, when detecting PSPB, each well or container of an assay may becoated with an antibody specific to PSPB. Each well or container may bepre-coated with the antibody before the coated well or container reachesthe tester.

Any appropriate assay or ELISA (sandwich, indirect, competitive,reverse, etc.) can be provided. The assay provided may be a polystyrenemicroplate, having wells/containers with inner surfaces capable of beingcoated with antibody. These inner surfaces may or may not be treatedwith substances known in the art to promote or enhance coating. Forexample the surface can be a maxisorp, Polysorp®, medisorp, Minisorp® orCovalink® surface. Each well or container may have a total surface areaof about 2.5 cm²/well. Each well or container may have a total volume ofabout 350 μl/well. Each well or container may have a suggested workingvolume of about 250 μl/well. Each well or container may be white oropaque to allow for easier visualization of any color, or any visuallydetectable change, occurring in or on the well or container. It will beappreciated that the size, surface area, total and/or working volumes,appearance, and/or color/visual parameters and/or qualities can bemodified as desired within the scope of the present disclosure. Forexample, the working volume for each well or container can be about 25μl/well to about 250 μl/well within the scope of the present disclosure.

In some embodiments, the test or support surface may be part of a vial(or container or well), a test strip, a chromatography substrate, a genechip, a Snap® test, or any other diagnostic test or test system used fordetecting markers, such as pregnancy markers. The test or supportsurface may be made of paper, plastic, glass, metal, etc. and takeseveral forms such as paddle, beads, wells, electrodes, etc.

In some embodiments, non-specific adsorption to the test surfaces coatedwith the BRE (e.g. the detection antibody), such as the coatedwell/container of an assay, may be minimized by blocking the testsurface with a blocking agent. The blocking agent may be one or moreproteins, sugars and/or polymers such as bovine serum albumin, gelatin,polyethylene glycol, sucrose, etc.

In some embodiments, the test surface coated with the BRE (e.g., thedetection antibody), such as the coated well/container of an assay, maybe coated with a preserving (or stabilizing) agent to preserve theactivity of the test surface. Test surfaces coated with the BRE and theblocking agent may also be coated with the preserving agent. Thepreserving agent may allow the test surfaces coated with the preservingagent, and the BRE and/or blocking agent, to be stored for an extendedperiod of time before use. Test surfaces coated with the preservingagent, and the BRE and/or blocking agent, may maintain immunologicalactivity for several months compared to if no preserving agent isemployed (where immunological activity of a test surface coated with theBRE and/or a blocking agent may continually decline over time).

The preserving agent may be composed of any organic or inorganic bufferwith some or all of the following characteristics: a concentrationbetween about 0.005 M and about 0.200 M, a pKa value between about 6.0and about 8.0, high solubility, non-toxicity, limited effect onbiochemical reactions, very low absorbance between about 240 nm andabout 700 nm, enzymatic and hydrolytic stability, minimal changes due totemperature and concentration, limited effects due to ionic or saltcomposition of the solution, limited interaction with mineral cations,and/or limited permeability of biological membranes. Stabilizationcompounds may be any reducing or non-reducing carbohydrate in the rangeof concentrations (weight/volume or volume/volume) from about 0.005% toabout 20%.

Preparation of Test Standards

There may be provided one or more test standards corresponding to one ormore fixed levels of the marker being detected. In some embodiments, afirst test standard and a second test standard may be provided. To setthe standard levels, the first test standard may correspond to a firstamount of the marker being detected. The second test standard maycorrespond to a second level of the marker being detected, which is adifferent level from the first amount.

When a pregnancy marker is being detected, each standard may correspondto a different fixed level of the pregnancy marker. The first or highstandard may correspond to the minimum level of the pregnancy markerfound in a pregnant animal. The second or low standard may correspond tothe maximum level of the pregnancy marker found in a non-pregnantanimal. The pregnancy marker used for the test standard(s) may be apurified, semi-purified or complex form of the marker of the speciesunder test or be from a species in which the cross-species of the markercan be used to set an accurate cutoff level for pregnancy statusdetermination.

For example, when the pregnancy marker being detected is PSPB, the highstandard may correspond to the level of PSPB corresponding to a minimumlevel of PSPB present in a pregnant animal. The high standard for aruminant may correspond to a concentration between about 0.01 ng/ml andabout 1.0 ng/ml of PSPB in serum. The low standard may correspond to thelevel of PSPB corresponding to the maximum level of PSPB present in anot pregnant animal. The low standard for a ruminant may correspond to aconcentration between about 0.2 ng/ml and about 0.4 ng/ml of PSPB inserum.

The high and low standards can be determined in a similar manner forwhatever pregnancy marker is being detected. For example, whenprogesterone is the pregnancy marker for a cow, then the high standardmay be about 2 ng/ml to 6 ng/ml of progesterone in serum and the lowstandard may be about 1 ng/ml to 2 ng/ml of progesterone in serum. Whenestrone sulfate is the pregnancy marker for a horse, then the highstandard may be about 13 ng/ml of estrone sulfate in serum and the lowstandard may be about 9.4 ng/ml of estrone sulfate in serum.

The one or more test standard(s) may be provided to a tester in multipleways. In some embodiments, a test kit, such as those in FIG. 2, may beprovided to the tester. The kit may include a reagent solution of thehigh standard S₁ and of the low standard S₂. The high and low standardsolutions may include fixed levels of the marker being detected, asdescribed above. The high and low test standards may also be preparedfrom any solution that can generate a signal (visual or otherwise)consistent respectively with a positive/reactive (e.g. pregnant) leveland a negative/non-reactive (e.g., not pregnant) level of the marker(s)being detected.

For an assay test, the high standard solution may be added to a firstwell or container of the assay and the low standard solution to a secondwell or container. Samples may then be tested in other wells orcontainers of the assay and compared against the low and high standards.In this way, the sample being tested may be compared in a similartesting environment (i.e., temperature, timing, preparation, etc.) asthe standards, which may generate more accurate results.

In some embodiments, the high and low test standards may be prepared andprovided to the tester in a ready-to-use condition for testing analysis.For example, when an assay is provided for performing the test, the highand low standards may be prepared and sealed in the first and secondwell or containers. A piece of paper may be included in the kitimprinted with signals (visual or otherwise) indicating the high and lowstandards levels. For example, a card may have color intensitiesimprinted on the card corresponding to the high and low standards, andthe sample color intensity can be compared to the cards. Containers orvials may be provided with the high and low standards already preparedfor comparison to the tested samples.

It will be appreciated that any of the above test standards can beprovided in lieu of, or in addition to, the other test standards.

Obtaining Sample(s) from Animal(s)

The type of sample retrieved from the animal may depend on the markerbeing detected. The sample may be a blood sample, such as of wholeblood, plasma, or serum. The sample may be of a biological or bodilyfluid, such as of saliva, reproductive tract secretions (i.e., vulva,vaginal, uterine, cervical, oviductal, etc.), and/or ectodermal or skinorigin secretions (e.g., tears, sweat, milk, urine, etc.). The samplemay be of a tissue, a cell, and/or of any biological solid. The samplemay be an extract of any of the above. The sample may be a combinationof any of the above.

Samples may be obtained from an animal using known retrieval proceduresspecific to the type of sample being taken. The time period in which thesample may be retrieved before testing may depend on the type of samplebeing obtained, the marker being detected, and/or the type of analysisor diagnosis being performed. The time period in which the sample shouldbe tested after being retrieved may depend on the type of sample taken,the marker being detected, and/or the type of analysis or diagnosisbeing performed.

For example, when detecting a pregnancy marker, one or more samples maybe retrieved from the tested animal after insemination of the animal.The time period for retrieving the sample may depend on the type ofanimal being tested, the pregnancy marker(s) being detected (e.g.,different markers appear at different stages of gestation in differentdetectable amounts), and/or the BREs being used to detect the pregnancymarker (e.g., an antibody selected could detect different variants ofpregnancy markers that appear at different stages of gestation indifferent detectable amounts).

As examples, in some embodiments, when detecting pregnancy of a ruminantusing PSPB as the marker, the sample may be retrieved about 30 days orlater after conception/insemination. When detecting pregnancy of a cowusing estrone sulfate as the marker, the sample may be retrieved about100 days or later after conception/insemination. When detectingpregnancy of a horse using estrone sulfate as the marker, the sample maybe retrieved about 70 days or later after conception/insemination. Whendetecting pregnancy of a cow using progesterone as the marker, thesample may be retrieved about 18 to about 24 days afterconception/insemination. The day of retrieving a sample can vary byspecies and may be near the time of expected heat if the animal has notconceived.

Testing Sample on Test Surface and Signal Development for Analysis

To detect if a marker is present in a sample, a signal from the samplemay be compared against the signals of a high standard and a lowstandard. A qualitative/visual signal may be generated or visualized ofthe sample and test standards for making the comparison. The visualindicator may visualize or generate a signal of the sample and standardshaving a magnitude corresponding to the level of the marker present. Thevisual indicator may visualize or generate a signal for the firststandard consistent with a first level of marker. The visual indicatormay visualize a signal for the second standard consistent with a secondlevel of marker.

For example, when detecting a pregnancy marker, the visual indicator mayvisualize for the high standard a signal consistent with a level, suchas the minimum level, of the pregnancy marker found in a pregnantanimal. The visual indicator may visualize for the low standard a signalconsistent with a level, such as the maximum level, of the pregnancymarker found in a not-pregnant animal. The magnitude of the signal froman animal sample generated by the visual indicator may be comparedagainst the standards to determine pregnancy.

Generating the visually detectable signal can be accomplished in severalways. Any visual indicator, including any dye, chromogen, substance,substrate, or solution capable of producing a qualitative indication orvisually detectable change may be utilized. The generated signal may bevisually detectable with or without special equipment. For example, thesignal may be a color change, or the generation of a color change alonga spectrum, that is visible without special equipment. In someembodiments, it is possible to detect changes in light absorbancevisually, with non-specialized light detection equipment, or specializedequipment (e.g., Spectrophotometer). In some embodiments, the signal maybe detected by measuring a change in a physical or chemical property ofthe substrate being tested based on the presence of a label, such as anenzyme label. Types of enzyme-labeled signals known to the art include:light absorbance, light emission, fluorescence, electrochemical signal,pH, etc.

As an example, FIG. 3 schematically illustrates embodiments of thepresent disclosure for generating a visually detectable signal of thesubstrate being tested. As shown in FIG. 3, test surfaces 26 a, 26 b,and 26 c may each be coated with a first antibody 28 specific to themarker 30 being detected. As shown in FIG. 4, which is discussed later,the coated test surfaces may be the inner surfaces of the wells orcontainers of an assay, such as a sandwich ELISA.

Returning again to FIG. 3, a high standard or control S₁ may be exposed(also referred to as introduced) to test surface 26 a, a low standard orcontrol S₂ may be exposed to test surface 26 b, and a sample S_(a) maybe exposed to test surface 26 c. Each may be exposed to test surfaces 26a, 26 b, 26 c under a substantially constant pressure and temperature,and for substantially the same time period. Each may be first exposed totest surfaces 26 a, 26 b, 26 c at substantially the same time. Duringthe exposure period, any marker 30 present in the standards and samplemay react and bind with the first detection antibody 28 coated on eachtest surface. At the conclusion of the exposure period, unbound standardor sample may be removed and discarded from each test surface.

The marker remaining bound to test surfaces 26 a, 26 b, 26 c may beconsistent with the level of marker present in the standards and sample.The more marker bound to the test surfaces, the more marker was present.

A signal corresponding to the bound marker may be visualized. Thegeneration of a signal from the bound marker may be accomplished byexposing to each surface 26 a, 26 b, 26 c a second detection antibody 32specific to the marker 30 being detected. The second detection antibody32 may be the same as, or different from, the first detection antibody28. The second detection antibody 32 may be conjugated or labeled (asindicated with a C in FIG. 3.). The second detection antibody 32 may beconjugated or labeled by the tester or before being provided to thetester. A conjugated or labeled second detection antibody may beprovided to the tester in a reagent solution R₁.

The second detection antibody 32 (or conjugated second detectionantibody) may be exposed to test surfaces 26 a, 26 b, 26 c under asubstantially constant pressure and temperature, and for substantiallythe same time period. The second detection antibody 32 (or conjugatedsecond detection antibody) may be first exposed to test surfaces 26 a,26 b, 26 c at substantially the same time. The second detection antibody32 (or conjugated second detection antibody) may be exposed to testsurfaces 26 a, 26 b, 26 c before, at the same time as, and/or after thesample and standards are exposed to test surfaces 26 a, 26 b, 26 c.During the exposure period, the second detection antibody 32 (orconjugated second detection antibody) may react with and bind to themarker 30 bound on each surface 26 a, 26 b, 26 c. At the conclusion ofthe exposure period, unbound second detection antibody 32 (or conjugatedsecond detection antibody) may be removed and discarded from testsurfaces 26 a, 26 b, 26 c.

The amount of second detection antibody 32 (or conjugated seconddetection antibody) bound to test surfaces 26 a, 26 b, 26 c may beconsistent with the level of marker 30 bound to each test surface. Themore second detection antibody 32 (or conjugated second detectionantibody) bound to each surface, the more marker 30 was bound to eachsurface.

To generate a visually detectable signal, the second detection antibody32 (or conjugated second detection antibody) may be labeled orunlabeled. If labeled, then the conjugated or labeled second detectionantibody 32 may generate a visually detectable signal upon binding tothe marker 30 on each test surface. If unlabeled, then additional orsecondary steps may be needed to label the second detection antibody 32(or conjugated second detection antibody) to generate a visuallydetectable signal.

FIG. 3 illustrates several examples of generating a visually detectablesignal of the second detection antibody 32 (or conjugated seconddetection antibody) bound to each test surface, which are discussedbelow.

a. Signal Development (if Conjugate (C)=Hapten): Hapten-Enzyme-VisualIndicator

In some embodiments, the second detection antibody 32 may be conjugatedwith a hapten, such as a small molecule. The hapten conjugated antibodymay act as a detector that detects the molecule of interest. The haptenmay be biotin, digoxigenin, dinitrophenol, fluroscein, etc. The seconddetection antibody conjugated with the hapten may be exposed to thebound marker 30 on each test surface 26 a, 26 b, 26 c as describedabove. Unbound second detection antibody conjugated with the hapten maybe removed and discarded from the test surfaces 26 a, 26 b, 26 c, asdescribed above.

An enzyme may be exposed to each test surface that may react with and/orbind to a hapten-conjugated detection antibody. The enzyme may behorseradish peroxidase, alkaline phosphatase, beta-galactosidase, or anyenzyme capable of reacting with and/or binding to a hapten-conjugatedantibody. The enzyme may be exposed to test surfaces 26 a, 26 b, 26 cunder a substantially constant pressure and temperature, and forsubstantially the same time period. The enzyme may be first exposed totest surfaces 26 a, 26 b, 26 c at substantially the same time. Duringthe exposure period, the enzyme may react with and/or bind to thehapten-conjugated second detection antibody 32 bound on each surface 26a, 26 b, 26 c. At the conclusion of the exposure period, unbound enzymemay be removed and discarded from test surfaces 26 a, 26 b, 26 c.

A visual indicator may be exposed to each test surface that may reactwith and/or bind to the selected enzyme. The visual indicator maygenerate a signal whose magnitude corresponds to the amount of enzymebound to each test surface. In some embodiments, the visual indicatormay be an enzyme substrate (e.g., 3, 3′, 5, 5′ tetramethylbenzidine(TMB)) specific to the selected enzyme (e.g., horseradish peroxidase).Any appropriate visual indicator can be used, such as TMB, fluroscein,etc. Other visual signal indicators may include X-gal, para-aminophenol,BCIP, p-nitrophenol, luminol, etc.

The visual indicator may be exposed to test surfaces 26 a, 26 b, 26 cunder a substantially constant pressure and temperature, and forsubstantially the same time period. The visual indicator may be firstexposed to test surfaces 26 a, 26 b, 26 c at substantially the sametime. During the exposure period, the visual indicator may react withand/or bind to the enzyme bound to each surface 26 a, 26 b, 26 c. Thereaction may generate a visually detectable change or signal. Therespective magnitude of the change or signal may correspond to theamount of marker 30 bound to each test surface 26 a, 26 b, 26 c. At thespecified time after the reaction begins, the signal may be detectedand/or observed with or without stopping the reaction.

In some embodiments, a hapten reactive molecule labeled with an enzyme(e.g. avidin-peroxidase, anti-digoxigenin-peroxidase) may be exposed toeach test surface that may react with and/or bind to a hapten-conjugateddetection antibody. The hapten reactive molecule may be labeled withhorseradish peroxidase, alkaline phosphatase, beta-galactosidase, or anyenzyme capable of reacting with and/or binding to a hapten-conjugatedantibody. The enzyme may be exposed to test surfaces 26 a, 26 b, 26 cunder a substantially constant pressure and temperature, and forsubstantially the same time period. The enzyme may be first exposed totest surfaces 26 a, 26 b, 26 c at substantially the same time. Duringthe exposure period, the enzyme may react with and/or bind to thehapten-conjugated second detection antibody 32 bound on each surface 26a, 26 b, 26 c. At the conclusion of the exposure period, unbound enzymemay be removed and discarded from test surfaces 26 a, 26 b, 26 c. Thelabeling with streptavidin-horseradish peroxidase and/oravidin-horseradish peroxidase may act as an enhancer that enhances thevisibility of the visually detectable color or signal. An enhancer, suchas strepavidin conjugated with horseradish peroxidase (SA-HRP) anddetected with 3,3′,5,5′-Tetramethylbenzidine (TMB), or stepavidinconjugated with alkaline phosphatase (SA-AP) and detected withp-nitrophenyl phosphate (pNPP), can be used as appropriate with any ofthe other embodiments disclosed in or within the scope of the presentdisclosure.

As shown in FIG. 2, the reagents and components necessary to perform thetest may be provided as Kit A. Kit A may include the test surfacescoated with the first detection antibody specific to the marker beingdetected, a bottle of reagent solution with high standard S₁, a bottleof reagent solution with low standard S₂, a bottle of reagent solutionR_(1a) with the hapten-conjugated second detection antibody, a bottle ofreagent solution R₂ with an enzyme, and a bottle of reagent solution R₃with the visual indicator.

b. Signal Development (if Conjugate (C)=Hapten): Hapten-Visual Indicator

The second detection antibody 32 conjugated with the hapten may beexposed to the bound marker 30 on each test surface 26 a, 26 b, 26 c asdescribed above. Unbound second detection antibody conjugated with thehapten may be removed and discarded from the test surfaces 26 a, 26 b,26 c, as described above.

A visual indicator may be exposed to the hapten-conjugated seconddetection antibody bound to each test surface 26 a, 26 b, 26 c. Thevisual indicator (e.g., TMB) may be any that is capable of reacting withand/or binding to a hapten-conjugated detection antibody in a mannerthat generates a visually detectable change or signal. The visualindicator may generate a change or signal whose magnitude corresponds tothe amount of hapten-conjugated second detection antibody bound to eachsurface 26 a, 26 b, 26 c.

The visual indicator may be exposed to test surfaces 26 a, 26 b, 26 cunder a substantially constant pressure and temperature, and forsubstantially the same time period. The visual indicator may be firstexposed to test surfaces 26 a, 26 b, 26 c at substantially the sametime. During the exposure period, the visual indicator may react withand/or bind to the hapten-conjugated second detection antibody 32 boundto each surface 26 a, 26 b, 26 c. The reaction may generate a visuallydetectable change or signal. The respective magnitude of the change orsignal may correspond to the amount of marker 30 bound to each testsurface 26 a, 26 b, 26 c. At the specified time after the reactionbegins, the signal may be detected and/or observed with or withoutstopping the reaction.

As shown in FIG. 2, the reagents and components necessary to perform thetest may be provided as Kit B. Kit B may include the test surfacescoated with the first detection antibody specific to the marker beingdetected, a bottle of reagent solution with high standard S₁, a bottleof reagent solution with low standard S₂, a bottle of reagent solutionR_(1a) with the hapten-conjugated second detection antibody, and abottle of reagent solution R₃ with the visual indicator.

c. Signal Development (If Conjugate (C)=Enzyme): Enzyme-Visual Indicator

As shown in FIG. 3, in some embodiments, the second detection antibody32 may be conjugated with an enzyme. The enzyme may be horseradishperoxidase, alkaline phosphatase, or any enzyme capable of reacting withand/or binding to marker 30.

The second detection antibody 32 conjugated with the enzyme may beexposed to the bound marker 30 on each test surface 26 a, 26 b, 26 c asdescribed above. Unbound second detection antibody conjugated with theenzyme may be removed and discarded from the test surfaces 26 a, 26 b,26 c, as described above.

A visual indicator may be exposed to each test surface 26 a, 26 b, 26 cthat reacts with and/or binds to the enzyme-conjugated second detectionantibody bound to each test surface. The visual indicator (e.g., TMB)may be any that is capable of reacting with and/or binding to anenzyme-conjugated detection antibody in a manner that generates avisually detectable change or signal. The visual indicator may generatea change or signal whose magnitude corresponds to the amount ofenzyme-conjugated second detection antibody bound to each surface 26 a,26 b, 26 c.

The visual indicator may be exposed to test surfaces 26 a, 26 b, 26 cunder a substantially constant pressure and temperature, and forsubstantially the same time period. The visual indicator may be firstexposed to test surfaces 26 a, 26 b, 26 c at substantially the sametime. During the exposure period, the visual indicator may react withand/or bind to the enzyme-conjugated second detection antibody bound toeach surface 26 a, 26 b, 26 c. The reaction may generate a visuallydetectable change or signal. The respective magnitude of the change orsignal may correspond to the amount of marker 30 bound to each testsurface 26 a, 26 b, 26 c. At the specified time after the reactionbegins, the signal may be detected and/or observed with or withoutstopping the reaction.

As shown in FIG. 2, the reagents and components necessary to perform thetest may be provided as Kit C. Kit C may include the test surfacescoated with the first detection antibody specific to the marker beingdetected, a bottle of reagent solution with high standard S₁, a bottleof reagent solution with low standard S₂, a bottle of reagent solutionR_(1b) with the enzyme-conjugated second detection antibody, and abottle of reagent solution R₃ with the visual indicator.

d. Signal Development (If Conjugate (C)=Visual Indicator): VisualIndicator

As shown in FIG. 3, in some embodiments, the second detection antibody32 may be labeled or conjugated with the visual indicator. The visualindicator may be any capable of producing a visually detectable changeor signal with or without special equipment. For example, the visualindicator may be an enzyme substrate that produces fluorescence that canbe detected by excitation using a light source. If the visual indicatoris a fluorescent marker, such as fluroscein, the signal may be developedusing light excitation of the fluorophore. Fluorescent signal detectionmay be accomplished by measuring the light emitted from the excitedfluorophore visually or with a light reader. The light reader may have afilter that is specific to the emitted wavelength of the fluorophorebeing detected.

The second detection antibody 32 conjugated with the visual indicatormay be exposed to each surface 26 a, 26 b, 26 c. The visualindicator-conjugated second detection antibody 32 may be exposed to testsurfaces 26 a, 26 b, 26 c under a substantially constant pressure andtemperature, and for substantially the same time period. The visualindicator-conjugated second detection antibody 32 may be first exposedto test surfaces 26 a, 26 b, 26 c at substantially the same time. Duringthe exposure period, the visual indicator-conjugated second detectionantibody 32 may react with and/or bind to the marker bound to eachsurface 26 a, 26 b, 26 c. The reaction may generate a visuallydetectable change or signal. The respective magnitude of the change orsignal may correspond to the amount of marker 30 bound to each testsurface 26 a, 26 b, 26 c. At the specified time after the reactionbegins, the signal may be detected and/or observed with or withoutstopping the reaction.

As shown in FIG. 2, the reagents and components necessary to perform thetest may be provided as Kit D. Kit D may include the test surfacescoated with the first detection antibody specific to the marker beingdetected, a bottle of reagent solution with high standard S₁, a bottleof reagent solution with low standard S₂, a bottle of reagent solutionR_(1c) with the visual indicator-conjugated second detection antibody.

e. Signal Development—Others

Several other visual indicators, and/or systems and methods, can beutilized to generate a visually detectable signal within the scope ofthe present disclosure. For example, any molecule capable of acting as avisual indicator may be used for signal development. The visualindicator may be or include fluorophors, lumiphors, upconvertingphosphors, dyes, chemiluminescent molecules, electrochemiluminescentmolecules, etc. These may be free, bound, conjugated to, or encapsulatedin nanoparticles. Other detection agents may be or include goldnanoparticles, silicon oxide nanoparticles, fluorescent nanoparticles;latex or other particles; etc. Other ways to generate a visual responsemay include using an enzyme-linked coagulation assay (see U.S. Pat. No.4,668,621); and/or an enzyme-linked precipitation assay. Other ways togenerate a visual response may include generating a detectableelectrical signal or refraction, such as by using enzyme-linkedamperometry.

As one example, a lateral flow strip or filter pad may include testingsurfaces coated with antibodies specific to the marker being detected.The high and low test standards may be exposed to a first and secondportion of the test surface. The standard may be exposed to a thirdportion of the test surface. As described above, any marker present inthe sample and/or standards may be bound to the test surface. Unboundsample and standards may be removed from the test surface. As describedabove, a second detection antibody specific to the marker being detectedmay be exposed and react with and/or bind to the marker bound on thetest surface. In some embodiments, unbound second detection antibody maybe discarded.

A visual indicator, such as a fluorescent nanoparticle, may be used forvisualizing the presence of the marker being detected on the testsurface. A fluorescent intensity may be created by excitation from alight source. A handheld reader of fluorescent intensity may be used toobserve the signal from the excitation. The magnitude of the fluorescentintensity may correspond with the level of second detection antibodybound to the marker on the test surface of the strip or pad. Themagnitude of the signal from the sample may be compared against the teststandards on the test surface. Additionally, or alternatively, the stripor pad may include an indication of intensities corresponding to the lowand high standards. An intensity visual discrimination card withindications corresponding to the high and low standards may also beprovided.

Analysis of Visually Generated Signal of Sample against Standards

As illustrated in FIG. 3, the visual indicator may give an indicationregarding the presence of the marker in the sample.

In some embodiments, the first (or high) standard may correspond to agreater magnitude of signal strength of the marker than does the second(or low) standard. The stronger the magnitude of the signal, the higherthe amount of marker that may be present in the sample. As such, if themagnitude of the sample signal is above (and in some cases equal to) thefirst (or high) standard, then that may indicate the sample is positiveor reactive. If the magnitude of the sample signal is below (and in somecases equal to) the second (or low) standard, than that may indicate thesample is negative or non-reactive. If the magnitude of the samplesignal is between the standards (and in some cases equal to the firststandard and/or second standard), then that may indicate the animalwhose sample is being tested should be rechecked or retested. A recheckor retest may indicate that another sample should be retrieved from theanimal for analysis at some time after the initial sample was retrievedfrom the animal for analysis. The later sample can be checked againstthe standards as above. For example, a sandwich ELISA may be configuredfor this type of analysis, and there are various other testing systemsthat can be configured for this type of analysis within the scope of thepresent disclosure.

In some embodiments, a greater amount of marker that may be present inthe sample may result in a decrease in the magnitude of the signal. Assuch, if the magnitude of the sample signal is below (and in some casesequal to) the first (or high) standard, then that may indicate thesample is positive or reactive. If the magnitude of the sample signal isabove (and in some cases equal to) the second (or low) standard, thanthat may indicate the sample is negative or non-reactive. If themagnitude of the sample signal is between the standards (and in somecases equal to the first standard and/or second standard), then that mayindicate the animal whose sample is being tested should be rechecked orretested. A recheck or retest may indicate that another sample should betaken from the animal for analysis at some time after the initial samplewas taken from the animal for analysis. The later sample can be checkedagainst the standards as above. For example, a competitive and/orsubstitute ELISA may be configured for this type of analysis, and thereare various other testing systems that can be configured for this typeof analysis within the scope of the present disclosure.

FIG. 4 is an example of signal analysis using the systems and methods ofthe present disclosure. As shown in FIG. 4, there may exist a spectrumof magnitude of reactivity level from 1 (low) to 5 (high). The firststandard S₁ may be the high standard and correspond to a reactivity of 4and the second standard S₂ may be the low standard and correspond to areactivity of 2. The signals of samples S_(a) may then be comparedagainst standards S₁ and S₂. In this example, samples S_(a) whose signalmagnitude is at or above the high standard, such as S_(a4) or S_(a5),may be considered positive or reactive. Samples S_(a) at or below thelow standard, such as S_(a1) or S_(a2), may be considered negative ornon-reactive. Samples S_(a) between the standards, such as S_(a3), maybe considered rechecks. In some embodiments, samples equaling the highstandard (e.g. S_(a4)) and/or low standard (e.g. S_(a2)) may instead beclassified as a recheck.

In some embodiments, the first standard S₁ may be the low standard andcorrespond to a reactivity of 4 and the second standard S₂ may be thehigh standard and correspond to a reactivity of 2. The signals ofsamples S_(a) may then be compared against standards S₁ and S₂. In thisexample, samples S_(a) whose signal magnitude is at or above the lowstandard, such as S_(a4) or S_(a5), may be considered negative ornon-reactive. Samples S_(a) at or below the high standard, such asS_(a1) or S_(a2), may be considered positive or reactive. Samples S_(a)between the standards, such as S_(a3), may be considered rechecks. Insome embodiments, samples equaling the low standard (e.g. S_(a4)) and/orhigh standard (e.g. S_(a2)) may instead be classified as a recheck.

It will also be appreciated that the samples S_(a) shown in FIG. 4 mayall be from the same animal, or from different animals. The samplesS_(a) may be a first sample of an animal (e.g. S_(a3)) and a recheckedsample of an animal (e.g. S_(a1) or S_(a5)) retrieved at some timeperiod after the first sample was retrieved.

Each magnitude 1 through 5 shown in FIG. 4 may correspond to differentcolors or to different levels of intensity along a spectrum for onecolor (e.g., from 1 low to 5 high). Each magnitude 1 through 5 maycorrespond to a mixture of color intensities of one or more differentcolors. Each magnitude 1 through 5 may correspond to a signal other thana color, such as a visually detectable signal.

FIG. 4 may represent the results of an assay test for detecting thepresence of a pregnancy marker (e.g. PSPB) in an animal sample todetermine pregnancy. The magnitude of the signal may representmagnitudes of a color, such as blue, that is present when the pregnancymarker may be present. The darker the color, the more pregnancy markermay be present. (As noted above, in some embodiments, this may bereversed such that the darker/higher the color intensity, the lesspregnancy marker may be present). The high test standard may be anintensity of blue color consistent with the level of the pregnancymarker found in a pregnant animal. The low test standard may be anintensity of blue color consistent with a level of the pregnancy markerfound in a not pregnant animal.

A qualitative, visual analysis may be performed to compare a sample inan assay well against the high and low test standards in other assaywells to determine if an animal is pregnant (equals blue color intensitysame or above high standard), not pregnant (equals blue color intensitysame or below low standard), or recheck (equals blue sample colorintensity between standards). Another sample can then be retrieved fromthe animal for analysis within a few days, perhaps within 7 days, iftesting a ruminant, after the first sample was retrieved that indicateda recheck.

In some embodiments, the use of an enhancer, such as those above, may beused to increase the intensity of the signal or color to give anincrease in the spread or dynamic range between the first and secondstandards. As an illustrative example, on an intensity scale, thelightest color may be a 1 and the darkest color may be a 2, with adynamic range between the colors of about 1 to 2. Use of the enhancermay increase the darkest color from 2 to 20 resulting in a dynamic rangeof 1 to 20, which is a 10-fold increase in the dynamic range. Use of theenhancer may therefore increase the intensity of the response for thehigh and low standards as well. The overall effect may be to give abetter separation between the low standard and the high standard. Thisbetter separation may allow for a tester to determine even more easilywhether a test sample is considered a positive, negative, or recheck bycomparing via a visual or qualitative analysis the signal of the sampleagainst the standards.

Other Advantages of the Systems and Methods of the Present Disclosure

The disadvantages of a binary test using blood-based markers is thetradeoff between sensitivity and specificity of the test depending uponthe selected cutoff level for the measured marker. This is an issue. Forexample, PSPB residual protein from a dead embryo may give a positivetest when in fact there is no pregnancy. Also, usually loss of an embryoearly in gestation, 30 to 80 days, can present results as a positivetest when in fact the cow is not pregnant.

Employing a three-result visual test (positive/reactive,negative/non-reactive, recheck) may avoid the sensitivity andspecificity issues associated with a two-result binary (positive,negative) test. For example, unlike in a binary test, a high sensitivitycan be maintained for a three-part test while the inefficiency of lostspecificity may be minimized by placing some or all potentially falsepositive, false negative and/or marginal/ambiguous result yields of abinary test into a recheck category. A sample categorized as recheckallows another sample to be taken at a later time, which may allow forcorrect identification (positive or negative) in the later test in aminimum amount of time.

Specifically for pregnancy testing in animals, the recheck category mayimprove management decision making. Normally, animals classified aspregnant will have a confirmation test up four to six weeks or moreafter the first test. If a binary test yields a false positive, thentime is wasted before re-insemination can occur because the falsepositive is unknown until the follow-up test.

Rather than yielding a false positive, the three-result pregnancy testmay be calibrated to place the false positive into a recheck category,but while maintaining a high sensitivity. Samples of animals in therecheck category may indicate that the animal needs to be retested forpregnancy at a date after the first test. Testing at a later time mayallow the pregnancy marker being detected to reach a testable level sothat testing a later sample from the animal may yield the correct “notpregnant” or “pregnant” result. The recheck date may be earlier than thedate of a standard follow-up procedure to confirm the results of thepregnancy test. Because the recheck can be performed onsite and visuallyusing the systems and methods of the present disclosure, there may betime and cost savings associated with the recheck category for pregnancymanagement.

The three-result test may allow for more effective administration ofhormones used to induce a new ovulation. Ovulation-inducing hormones areoften administered after determining an animal is not pregnant. Thehormones may allow the animal to return to heat and be mated againwithin a few days after the not pregnant determination. However, if abinary test yields a false negative for an animal that is otherwisepregnant, then the hormones administered to the animal may induce anabortion of a viable fetus. Allowing a classification of these falsenegatives as a recheck may allow follow-up testing within a week of therecheck result. As such, the false negative of a binary test and inducedabortion may be avoided, saving time and money.

Another advantage of the recheck category may be physiologicaladvantages associated with using a recheck category. For example, thereare several physiological advantages to the recheck category when thepregnancy marker being detected is PSPB. One advantage is that, if anembryo dies, PSPB will stop being produced by the placenta but therewill be residual PSPB in the blood. This is true no matter what form ofPSPB molecule is being detected. The recheck category may be calibratedso that a residual level of PSPB in the blood indicates a retest. ThePSPB will eventually clear from the blood and reach a non-detectablelevel. Taking a sample later for the recheck would likely indicate thatthe PSPB is below the cutoff and the animal is not pregnant followingdeath of the embryo.

For example, if an animal loses an embryo anytime between about 30 daysto about 75 days after insemination, it will take about 3 days to about6 days for PSPB to clear from the blood. This is because the PSPBhalf-life in maternal serum is about 3 to 7 days, depending upon thevariant of the PSPB molecule. If the high and low standards are properlycalibrated to account for this, an initial check of a sample takenwithin this clearance time may indicate a recheck. For example, taking asample 3 to 7 days after the initial sample was taken may be outsidethis clearance time and may indicate a not pregnant result.

Another physiological advantage is that the test will indicate a retestfor some slowly developing embryos that are not producing enough PSPB ataround 30 days-post insemination as to properly indicate whether theruminant is pregnant. During the later retest, the result may indicatethe ruminant is not pregnant if the embryo dies, or pregnant if theembryo grows.

Other advantages include that if a sample is withdrawn from a ruminantduring the post-partum period when residual PSPB is present, then thetest may indicate a recheck. If a sample is withdrawn from a ruminantduring the post-partum period when residual PSPB is present, andnew-embryo PSPB is present in low amounts, the test may also indicate arecheck. The later test can indicate correctly whether or not theruminant is pregnant.

To achieve the above advantages for a three-result pregnancy testdetecting PSPB, the high standard signal may be shifted lower to placefewer pregnant cows in the recheck category while still capturing alarge percentage of the overlapping not pregnant cows as a recheck. Assuch, and just as an example, it may be possible to categorize duringthe initial check approximately 99% of the pregnant cows as pregnant and90% of the not pregnant cows as not pregnant. The remaining cows may becategorized as a recheck. Those categorized as a recheck may be retestedwithin a week with greater than 98% of the rechecked samples correctlybeing categorized as pregnant or not pregnant.

A three-result onsite, visual test may allow for achieving a higherspecificity and sensitivity in a shorter testing period than possiblewith a two-result binary test.

Validation of Properly-Functioning Test

Methods may be employed to ensure that the test is properly calibratedand/or working. As an example, the high and low standards may be used asan initial check to determine if the test is properly calibrated and/orworking. For example, the magnitude of the reaction (e.g., the signal orcolor intensity) for the high standard should be more intense than forthe low standard. (The magnitude may be reversed in some embodimentssuch that the low standard may have a more intense signal or color thanthe high standard). If not, the test is not working properly. If thehigh and low standards look the same (e.g. signal or color intensity issame), then that may indicate the test is not working properly.

A test kit (such as Kits A-D in FIG. 2) may include a card with anindicia matching the magnitude of intensity (e.g., signal or colorintensity) of the standards. If the intensity of the standards does notmatch the intensity on the color card, then the test may not be workingproperly.

The test kit may include (in addition to standards) containers ofreagent solution with different sera of an animal that yield a testresult of not pregnant and/or recheck and/or pregnant for the animal.These sera can be used to validate if the test yields the proper resultfor each sera and may therefore be functioning properly. One or acombination of the above can be used for validating if the test isworking properly.

EXAMPLES

The following examples are included as non-exclusive, illustrativeembodiments of the present disclosure. Those of skill in the art should,in light of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe present disclosure

Example A Development of Reagents for ELISA

Bovine PSPB (bPSPB) was isolated after the methods of Hamilton (1979),Butler (1980), and Butler et al., (1982) from placenta of cows that wereless than 100 days in gestation; more specifically, they were from 25 toless than 100 days in gestation. The isolated PSPB was used to makestandards. The high standard was made by diluting the PSPB to aconcentration of 0.2 ng ml⁻¹ in steer serum or virgin heifer serum(VHS). The low standard was made by diluting the PSPB to a concentrationof 0.1 ng ml⁻¹ in steer serum or VHS.

Additionally, PSPB was conjugated to tetanus toxoid and was used toimmunize New Zealand White rabbits (Sasser et al., 1986). Anti-serum wascollected and tested for reactivity to the bPSPB standards. A portion ofthe rabbit anti-serum was used for IgG isolation using a Protein Gcolumn (Pierce). The tetanus toxoid-conjugated PSPB was also used toimmunize mice and develop monoclonal antibodies using procedures knownto the art. These standards and antisera were used to develop variousPSPB ELISA tests for pregnancy in livestock.

Ovine PSPB was isolated after the methods of Willard et al., 1995.Antisera against ovine PSPB was prepared as in Sasser et al., 1986.These reagents were used in the development of the ovine and caprineELISA.

Buffers, chemicals and reagents were developed and used in a mannerfamiliar to those skilled in the art of ELISA test development.

It will be appreciated that the development of the reagents, antibodiesand standards for use with a cELISA for measuring estrone sulfate orprogesterone can be developed in a manner familiar to those skilled inthe art of ELISA test development.

Example B Determination of Recheck Levels for PSPB

A test yielding pregnant, non-pregnant, and recheck cutoffs wasdeveloped based upon the radioimmunoassay (RIA) data of Sasser et al.,1986. Biol Reprod and Noyes et al., 1997. In the radioimmunoassay, acutoff of 95 or 93% of buffer control of the binding of radio-iodinatedPSPB was the inhibition required to reach a call of pregnancy for thetest sample. Any lower percentages are all called pregnant. A percentageabove 95 or 93% resulted in a non-pregnancy call. The nanogram amount ofPSPB that gives a 93% radioimmunoassay cutoff was used as the cutoff inthe ELISA plate test. It must be appreciated that protein isolates ofPSPB protein fractions will have different specific activities (PSPBprotein/total protein content of the preparation) between batches. Thenanogram amount of high standard and low standard required in an assayto develop a cutoff must be tested against field samples from animals tolearn the nanogram amount to use for the low and high standard. Knowingthe pregnancy status of those animals at the preferred time of testingis necessary to assure that the nanogram amounts apply to the practiceof blood-based pregnancy testing. Experience in use of the ELISA incattle testing showed that a recheck range was needed in order tocapture cattle that had PSPB in serum but at a level that warrantedfurther testing to assure pregnancy or non-pregnancy.

These standard amounts provide guidelines for color development in avisual test for PSPB or other pregnancy markers. As an example for PSPB,a strong color indicates binding and substrate reactivity of theHRP-labeled antibody conjugate to the bound PSPB, and is a positiveindication of pregnancy as would be the case, for example, in pregnantanimals that are 30 days post breeding. Weak color development indicateslittle or no binding of the HRP-labeled antibody conjugate due to theabsence of PSPB in the sample, and is a negative indication ofpregnancy. Color development between standards indicates a recheck.Color for a recheck status could be indicative of embryonic death inpreviously pregnant cows; a value in late postpartum cows; a value incows that were sampled too early in pregnancy; or a value, in rarecases, of cows with slow growing embryos. Standards of knownconcentrations of PSPB or other markers for pregnancy are run with eachassay and are used to determine the visual color values for assigningpregnant, non-pregnant and recheck status.

Example C ELISA for PSPB Detection Onsite with a Visual Result

The purified rabbit IgG from the Protein G column was labeled withbiotin using an NHS-LC-Biotin labeling kit (Pierce). Following removalof excess biotin using a molecular weight cutoff spin column, the biotinlabeled IgG (Biotin IgG) was diluted 1:2 in Stabilzyme stabilizersolution (SurModics, Inc) and further diluted to a final dilution of1:2000 in Tris Buffered Saline with 1% bovine serum albumin and 0.05%TWEEN-20 polysorbate 20 (TBSTB) pH 7.4, and stored at 4 degrees Celsius.

Horseradish peroxidase-labeled Streptavidin (SA-HRP) was purchased fromPierce, diluted 1:100 in Stabilzyme stabilizer solution (SurModics, Inc)and further diluted to a final dilution of 1:5000 in TBSTB and stored at4 degrees Celsius.

A NUNC C8 maxisorp strip well micro-titre clear or white polystyrenetest plates were used for all tests (NUNC, Inc.). Maxisorp strip plateswere coated with 150 microliters of anti-PSPB polyclonal rabbitanti-sera diluted 1:25000 in 0.1 M sodium carbonate buffer pH 9.6 for 18hours at room temperature with constant agitation. Following coating,the solution was removed from the plates and 200 microliters of 0.5%bovine serum albumin in 0.1 sodium carbonate buffer pH 9.6 was added toeach well and allowed to react for 15 minutes at room temperature withconstant agitation. The plates were then washed four times with 300microliters of Tris buffered saline with 0.05% TWEEN-20 polysorbate 20(TBST), blotted dry on a paper towel, covered with parafilm and placedat 4 degree Celsius.

Before performing the assay on test samples, the coated strip plates,standards, Biotin IgG, SA-HRP, test samples, and buffers were allowed toequilibrate to room temperature. The appropriate number of strip wellswere used to accommodate the number of test samples being tested, plustwo wells for the high and low standards.

The strip wells were initially wetted with 40 microliters of samplebuffer (80 mM sodium phosphate, 265 mM sodium chloride, 40 mM EDTA,0.05% Proclin, 0.4% gelatin, 0.02% TWEEN-20 polysorbate 20, and aserum-based blocking agent (e.g., 5-15% of normal cow, deer, goat,mouse, rabbit, sheep, wild ruminant serum, etc.) using a drop bottle(one drop). A transfer pipette was then used to place 120 microliters(two drops) of the high standard in the upper left most well. Two dropsof the low standard were placed in the well immediately below the highstandard using a different transfer pipette. Each successive sample wasadded to the plate by transferring two drops with a new transferpipette. The position of each sample was recorded on a grid consistingof 96 squares that represent the wells in a 96-well plate. The plate wasgently swirled and allowed to incubate at room temperature on the benchtop for 30 minutes. Following the incubation, the plate was emptied bypouring the samples out of the wells and lightly blotted on a papertowel. A squirt bottle was used to wash each well by filling the wellswith TBST followed by removal of the solution and blotting on a papertowel. The washing was performed four times.

A dropper bottle was then used to add four drops (160 ul) of Biotin IgG(1:2000 in TBSTB) to each well. The plate was gently swirled and allowedto incubate at room temperature on the bench top for 15 minutes.Following the incubation, the plate was emptied by pouring the samplesout of the wells and lightly blotted on a paper towel. A squirt bottlewas used to wash each well by filling the wells with TBST followed byremoval of the solution and blotting on a paper towel. The washing wasperformed four times.

A dropper bottle was then used to add four drops (160 ul) of SA-HRP(1:5000 in TBSTB from a 1 mg/ml stock solution) to each well. The platewas gently swirled and allowed to incubate at room temperature on thebench top for 15 minutes. Following the incubation, the plate wasemptied by pouring the samples out of the wells and lightly blotted on apaper towel. A squirt bottle was used to wash each well by filling thewells with TBST followed by removal of the solution and blotting on apaper towel. The washing was performed four times.

Four drops of TMB Max (Neogen Inc.) were added to each well using adropper bottle. The enzymatic reaction between the HRP and TMB wasallowed to proceed for five minutes. During this time, a blue colorbecame apparent in the standard wells and samples that contained PSPB.At five minutes, a dropper bottle was used to add 1 drop (40microliters) of 2% sodium fluoride to each well.

Each well was then compared visually with the high and low standardwells. Samples that achieved a signal that was equal to or greater thanthe high standard were categorized as pregnant. Samples with a signalless than the low standard were categorized as not pregnant. Samplesthat had signals that fall between the two standards were categorized asrecheck.

It is to be appreciated that ovine, caprine, cervid and other domesticand wild ruminant animals can be tested for pregnancy by this method ofvisual detection of the test sample.

Pregnancy status categorization of cows using these standards wasconfirmed using a commercially available laboratory assay of BioTrackingLLC. In the confirmation assay, the optical density for each well wasobtained from a plate reader with a 650 nanometer filter before stoppingthe enzymatic reaction (VersaMax, Molecular Devices, Inc). The enzymaticreactions were stopped with 1M sulfuric acid and read using a platereader with a 450 nanometer filter (VersaMax, Molecular Devices, Inc).

Example D Use of Various Visual Classification in Animal Management

As embryos and placenta grows, there is an increase in development ofbinucleated cells within the trophoblastic ectoderm of the conceptus.These cells are the source of PSPB in the maternal blood (Eckblad etal., 1985). As time progresses from conception to 30 days in gestation,the concentration of PSPB in blood increases from non-detectable levelsto fully detectable levels. Some cows have PSPB in serum beginning at 15days after breeding while all have it by 28 (Sasser et al., 1986) to 30(Humblot et al. 1988) days. The BioPRYN test is used to detect pregnancyfrom 30 days until term pregnancy (Howard et al, 2007). Likewise, if anembryo dies after there is a detectable level of PSPB in serum of themother then PSPB will decline to non-detectable levels. The half-life ofPSPB in serum is approximately 7 days in postpartum cows (Kirakofe etal., 1993) or cows in which embryonic death was induced by infection ofthe uterus (Semambo et al., 1992).

During either the incline of embryo growth or decline after embryo deathor parturition, there is a period of time in which marginal levels ofPSPB are present in serum. This results in uncertainty in prediction ofpregnancy status leading to placing cows in the recheck category. AnELISA assay that detects PSPB in bovine sera binds PSPB to antibodies(anti-PSPB) coated in the wells and PSPB is detected by secondarybinding of horseradish peroxidase (HRP)-labeled anti-PSPB antibodies.Binding of the HRP-labeled antibody conjugate is detected by theaddition of 3,3′,5,5′-tetramethylbenzidine (TMB) and is quantified bythe subsequent color development. A strong color indicates binding andsubstrate reactivity of the HRP-labeled antibody conjugate to the boundPSPB, and is a positive indication of pregnancy as would be the case,for example, in pregnant animals that are 30 days post breeding. Weakcolor development indicates little or no binding of the HRP-labeledantibody conjugate due to the absence of PSPB in the sample, and is anegative indication of pregnancy. Color development between standardsindicates a recheck. Color for a recheck status could be indicative ofembryonic death in previously pregnant cows; a value in late postpartumcows; a value in cows that were sampled too early in pregnancy; or avalue, in rare cases, of cows with slow growing embryos. Standards ofknown concentrations of PSPB are run with each assay and are used todetermine the visual color values for assigning pregnant/not pregnantand recheck status.

Over time, the level of PSPB in sera of cows with early growing embryoswill change over time. The levels of PSPB in sera of cows during thepostpartum period; cows will transition from a high degree of color fora positive postpartum cow to a moderate color for a recheck postpartumcow to a low or no color for non-pregnant postpartum cow or cows withembryonic death. Cows are known to transition to the non-pregnant statusbetween 50 and 90 days postpartum (Kirakofe et al. 1993).

The economic value of a visual recheck category exists. For example, ifa cow is 30 days in gestation and the test reveals a recheck, the cowcan be tested within 3 to 7 days to confirm the status because the PSPBvalue will either increase into the pregnancy status or decline into thenon pregnancy status. Within this time period, 87 percent of the recheckcows will likely be not pregnant on the next test while 13 percent willbe pregnant (Biotracking LLC, unpublished). Under the conventionalrectal palpation method, these cows would be tested again at 60 to 80days after breeding and time in being non-pregnant is extended. There istherefore considerable cost and time savings to the farming operation byusing an onsite test employing the visual recheck. Likewise, rectalpalpation is not done until 35 or later after breeding. Finding thenon-pregnant cows earlier with PSPB analysis using an onsite testemploying the visual recheck saves time and money in learning of thenon-pregnant cows. These cows can be treated with hormones to initiateheat and re-breed immediately. Thus the recheck category coupled withearly testing is extremely valuable to management of the animals.

Additionally, using the recheck status by visual color development in atest allows for more clearly identifying the non-pregnant cow at anearlier time. Placing cows into the Recheck category allowed for safeidentification of pregnant cows as early as 28 days after Al. Sevenpercent of pregnant cows were placed in the non-pregnant category on Day26. By Day 28 none of the pregnant cows were so placed while on this daysome were in the Recheck category. Thus management can safely treatthose categorized as non-pregnant without aborting any embryos. They canalso retest the recheck cows within a week after the previous testing tospecifically categorize the pregnancy status. This retest saves time inre-breeding non-pregnant cows. Additionally, it is possible to placemore animals in the recheck rather than non-pregnant category bylowering the visible cutoff for the recheck range. This would allowearlier testing and capture, as recheck, more animals classified asnon-pregnant such that they will be tested again within a week to betteridentify the status. Many of these will end up in the pregnant categorybecause they were low in PSPB because of early testing, not embryonicdeath. This modification of the recheck category allows to test earlierand identify non-pregnant cow for early re-breeding.

Optical density is an indicator of visual color and a measure of PSPB inserum of cows, sampled twice daily, that were pregnant or not pregnant.The Repeat Range is an OD value that indicates that the cow must betested again to get a definitive result. Within one to two days, the ODprogressed from low color to a color indicative of pregnancy. If anembryo has died at 28 days after AI, the OD would decline again throughthe Repeat Range and then to the non-pregnant range. In groups of cows,there will be those in the Repeat category due to delayed embryo growthor early testing or embryo death.

Example E Ovine (Sheep)/Caprine (Goat) Pregnancy ELISA Assay for theLaboratory

This antigen-capture, or “sandwich,” ELISA detects PSPB in ovine orcaprine sera. Serum PSPB binds to antibodies coated in the wells and isdetected by secondary binding of a labeled antibody. Binding of thelabeled antibody conjugate is detected by the addition of the enhancer(e.g. SA-HRP) and 3,3′,5,5′-tetramethylbenzidine (TMB) and is quantifiedby the subsequent color development. A strong color indicates bindingand substrate reactivity of the labeled antibody conjugate to the boundPSPB, and is a positive indication of pregnancy. Weak color developmentindicates little or no binding of the labeled antibody conjugate due tothe absence of PSPB in the sample, and is a negative indication ofpregnancy. Color development between the standards indicates a recheck.Standards of known concentrations of PSPB are run with each assay andare used to determine the optical density values for assigningPregnant/Not Pregnant ranges.

Components of the Assay Levels A Antibody coated plates 5 B SG SampleBuffer  30 ml C Open Goat Serum (OGS) None D PSPB Standard: HighStandard  9 ml E PSPB Standard: Low Standard  9 ml F Pregnant Goat Serum(PGS) None G SG Conjugate Buffer 250 ml H B6 rabbit α-bovine PSPBconjugate 750 μl I Enhancer (e.g., SA-HRP) 400 μl J 20X Wash Bufferconcentrate 120 ml K 250X TMB Substrate Solution  1.5 ml LPhosphate-Citrate Buffer 125 ml

Other Materials for Performing Test

Single and multichannel adjustable volume pipettors and disposable tips.Multichannel pipettor reservoirs. Microplate reader with the capabilityto read at 650 (or 630) and 450 nm. Deionized or distilled water.Graduated cylinders and beakers. Manual or automatic plate washer.Timer. Platform shaker and shaker/incubator. Microtiter plate covers. 3%hydrogen peroxide. Stop solution (1 M H₂SO₄).

Storage and Stability

Store all reagents except for the 20× wash buffer (J) at 4° C. The washbuffer concentrate must be stored at room temperature. Reagents willremain stable when stored as indicated.

Assay Procedure

1. Warm up all kit reagents, plates and samples.

2. Prepare the plates (A). Add 50 μl of SG Sample Buffer (B) (SG Samplebuffer can be 80 mM sodium phosphate, 265 mM sodium chloride, 80 mMEDTA, 0.05% Proclin, 0.4% gelatin, 0.02% TWEEN-20 polysorbate 20, and aserum-based blocking agent (e.g., 5-15% of normal cow, deer, goat, mice,rabbit, sheet, wild ruminant serum, etc.) to each well.

3. Add controls and samples. Run 150 μl of the controls provided withthe kit as follows. Use a fresh tip for each solution:

Well A-1: OGS (Open Goat Serum) (C) Wells B, C, D-1 PSPB Standard: HighStandard (D) Wells E, F, G-1 PSPB Standard: Low Standard (E) Well H-1:PGS (Pregnant Goat Serum) (F)

Add 150 μl of serum to the remaining wells. Use a fresh tip for eachsample.

4. Incubate the plates. Seal the wells with ParaFilm or DuraSeal, coverwith a microplate lid, and incubate overnight at room temperature withshaking.

5. Prepare the Conjugate. Prepare 1× antibody conjugate by diluting 2parts of the B6 conjugate (H) with 998 parts of SG Conjugate Buffer (G)(SG Conjugate Buffer can be 40 mM sodium phosphate, 132.5 mM sodiumchloride, 20 mM EDTA, 0.05% Proclin, 0.2% gelatin, 0.01% TWEEN-20polysorbate 20, and a serum-based blocking agent (e.g., 5-15% of normalcow, deer, goat, mice, rabbit, sheet, wild ruminant serum, etc.). For 96wells, mix 40 μl of the B6 conjugate (H) with 20 ml of SG ConjugateBuffer (G).

6. Prepare the Wash Solution. Prepare 1× Wash Buffer by diluting onepart of the 20× Wash Buffer Concentrate (J) (20× Wash Buffer Concentratecan be 200 mM sodium phosphate, 2.4 M sodium chloride, 0.5% TWEEN-20polysorbate 20) with 19 parts of deionized or distilled water.Approximately 3 ml/well of 1× wash solution will be required for thiswash step and the wash step below for a total of 9 washes.

7. Wash wells 4 times: After the overnight incubation, remove theParaFilm or DuraShield and wash the plate four times. Save theDuraShield for the next incubation.

If an automatic washer is used, place the plate on the washer and wash 4times with a volume of 300 μl. Set the washer to soak for 10 seconds andaspirate for 4 seconds between each wash. Remove any residual washsolution by striking the inverted plate on a paper towel (blot dry). Ifmanual washing is used, dump the contents of the wells into a sink ortub and then blot dry. Using a multichannel pipettor or Repeat Pipettor,add 300 μl of 1× wash buffer to each well and swirl the plate for 10seconds. Repeat the washing procedure 3 more times (4 washes total).

8. Add Conjugate: Using a single- or multi-channel pipettor, add 200 μlof the conjugate from step 5 to each well. Cover with fresh ParaFilm orthe DuraSeal from Step 4, and incubate at 37° C. and 100 RPM for 60minutes.

9. Prepare Enhancer. Prepare 1× Enhancer by diluting 1.25 parts of theEnhancer (I) with 998.75 parts of SG Conjugate Buffer (G). For 96 wells,mix 25 μl of the Enhancer (I) with 20 ml of QUICK Buffer (G) (QuickBuffer can be phosphate buffered saline pH 7.2 with 0.5% bovine serumalbumin and 0.05% TWEEN-20 polysorbate 20).

10. Dump, blot and wash the wells 1 time. After the 60 minute incubationlisted in Step 8, dump out the conjugate solution and blot dry on apaper towel. Wash the plate one time with 1× wash buffer. Dump out thewash solution and blot dry on a paper towel.

11. Add Enhancer. Using a single- or multi-channel pipettor, add 200 μlof the conjugate from step 9 to each well. Cover with fresh ParaFilm orthe DuraSeal from Step 4, and incubate at 37° C. with shaking for 60minutes.

12. Wash wells 4 times: After the 60 minute incubation listed in Step11, repeat the washing procedure described in Step 7, using theremaining 1× wash buffer (4 washes total).

13. Prepare the substrate solution: Prepare 1×TMB substrate by dilutingone part of the 250×TMB Substrate solution (K) with 250 parts ofPhosphate-Citrate Buffer (L) (Phosphate citrate buffer can be 50 mMsodium phosphate adjusted to pH 4.5 with citric acid). It is notnecessary to adjust for the slight volume change. For 96 wells (20 mltotal volume), add 80 μl of TMB Substrate Solution (K) to 20 ml ofPhosphate-Citrate Buffer (L) and mix well by swirling in a beaker. Add40 μl of 3% hydrogen peroxide and mix well.

14. Add substrate solution: Add 200 μl of the substrate solutionprepared in Step 13 to each well. After all of the wells have beenfilled, gently mix by swirling a few times in each direction. Incubateat room temperature for 15 minutes, swirl the plate for 5 seconds, andimmediately read the plate at 650 or 630 nm. If the average OD of thehigh standards is not approximately 0.12, continue the incubation for 2additional minutes and repeat the reading. When the average OD of thehigh standards is between 0.12 and 0.15, add the stop solution.

15. Add the Stop Solution: Add 100 μl of the stop solution (1M H₂SO₄) toeach well. After all of the wells have been filled, gently mix the wellcontents by swirling a few times in each direction. Add the stopsolution in the same order and timing as was done for the TMB.

16. Read the plate: Within 30 minutes after adding the stop solution,the plate should be read on a plate reader at 450 nm. For MolecularDevices scanners running SOFTmax software, export the reading as a textfile for data analysis.

***NOTE: This test is time dependent and assumes loading of a platetakes less than 30 minutes at room temperature. If the sample loadingprocedure takes longer than this prescribed time, or if loading multipleplates, pre-plate and transfer samples and standards with amulti-channel pipettor using a new tip for each sample.

Example F Quick Test: Bovine Pregnancy ELISA Assay

This antigen-capture, or “sandwich,” ELISA detects PSPB in bovine sera.Serum PSPB binds to antibodies coated in the wells and is detected bysecondary binding of a labeled antibody. Binding of the labeled antibodyconjugate is detected by the addition of the enhancer (e.g. SA-HRP) and3,3′,5,5′-tetramethylbenzidine (TMB) and is quantified by the subsequentcolor development. A strong color indicates binding and substratereactivity of the labeled antibody conjugate to the bound PSPB, and is apositive indication of pregnancy. Weak color development indicateslittle or no binding of the labeled antibody conjugate due to theabsence of PSPB in the sample, and is a negative indication ofpregnancy. Color development between the standards indicates a recheck.Standards of known concentrations of PSPB are run with each assay andare used to determine the optical density values for assigningPregnant/Not Pregnant ranges.

Components of the Assay Amount A Antibody coated plates 3 B SampleBuffer 1 bottle C Virgin Heifer Serum (VHS) 2 vials D PSPB Standard:QUICK Hi Std 1 vial E PSPB Standard: QUICK Lo Std 1 vial F Pregnant CowSerum (PCS) 2 vials G QUICK Buffer 1 bottle H Detector concentrate 1vial I Enhancer concentrate (SA-HRP) 1 vial J 20X Wash Bufferconcentrate 1 bottle K TMB Substrate concentrate 1 vial L QUICKPhosphate-Citrate Buffer 1 bottle

Other Materials Needed for Test

Single and multichannel adjustable volume pipettors and disposable tips.Multichannel pipettor reservoirs. Microplate reader with the capabilityto read at 650 (or 630) and 450 nm. Deionized or distilled water.Graduated cylinders and beakers. Manual or automatic plate washer.Timer. Microtiter plate covers. 3% hydrogen peroxide. Stop solution (1 MH₂SO₄).

Storage and Stability

Store all reagents except for the 20× wash buffer (J) at 4° C. The washbuffer concentrate must be stored at room temperature. Reagents willremain stable when stored as indicated.

Assay Procedure

Pre-plate by aliquoting serum samples to an empty micro-titer plate whenassaying large numbers of samples

1. Warm up all kit reagents, plates and samples.

2. Prepare the plates (A). Add 50 μl of Sample Buffer (B) to each well.

3. Add controls and samples. Run 150 μl of the controls provided withthe kit as follows. Use a fresh tip for each solution:

Well A-1: VHS (Virgin Heifer Serum) (C) Wells B, C, D-1 PSPB Standard:High Standard (D) Wells E, F, G-1 PSPB Standard: Low Standard (E) WellH-1: PCS (Pregnant Cow Serum) (F)

Add 150 μl of serum to the remaining wells. Use a fresh tip for eachsample.

4. Incubate the plates. Seal the wells with ParaFilm or DuraSeal, coverwith a microplate lid, and incubate for one hour at room temperature (ator near 70 degrees Fahrenheit).

5. Prepare the Detector Solution. Prepare 1× Detector solution bydiluting 6 parts of the Detector concentrate (H) with 994 parts of QUICKBuffer (G) (Quick Buffer can be phosphate buffered saline pH 7.2 with0.5% bovine serum albumin and 0.05% TWEEN-20 polysorbate 20). For 96wells, mix 120 μl of the Detector (H) with 20 ml of QUICK Buffer (G).

6. Prepare the Wash Solution. Prepare 1× Wash Buffer by diluting onepart of the 20× Wash Buffer Concentrate (J) with 19 parts of deionizedor distilled water. Approximately 3 ml/well of 1× wash solution will berequired for this wash step and the wash step below for a total of 9total washes.

7. Wash wells 4 times: After the one hour incubation, remove theParaFilm or DuraShield and wash the plate four times. Save theDuraShield for the next incubation. If using more than one plate dumpall plates at the same time to keep timing consistent.

If an automatic washer is used, place the plate on the washer and wash 4times with a volume of 300 μl. Set the washer to soak for 10 seconds andaspirate for 4 seconds between each wash. Remove any residual washsolution by striking the inverted plate on a paper towel (blot dry). Ifmanual washing is used, dump the contents of the wells into a sink ortub and then blot dry. Using a multichannel pipettor or RepeatPipettor,add 300 μl of 1× wash buffer to each well and swirl the plate for 10seconds. Repeat the washing procedure 3 more times (4 washes total).

8. Add Detector Solution: Using a single- or multi-channel pipettor, add200 μl of the conjugate from Step 5 to each well. Cover with freshParaFilm or the DuraSeal from Step 4, and incubate at room temperaturefor one hour.

9. Prepare Enhancer solution. Prepare 1× Enhancer solution by diluting 2parts of the Enhancer concentrate (I) with 998 parts of QUICK Buffer(G). For 96 wells, mix 40 μl of the Enhancer (I) with 20 ml of QUICKBuffer (G).

10. Dump, blot and wash the wells 1 time. After the one hour incubationlisted in Step 8, dump out the conjugate solution and blot dry on apaper towel. Wash the plate one time with 1× wash buffer. Dump out thewash solution and blot dry on a paper towel.

11. Add Enhancer solution. Using a single- or multi-channel pipettor,add 200 μl of the conjugate from step 9 to each well. Cover with freshParaFilm or the DuraSeal from Step 4, and incubate at room temperatureone hour.

12. Wash wells 4 times: After the one hour incubation listed in Step 11,repeat the washing procedure described in Step 7, using the remaining 1×wash buffer (4 washes total).

13. Prepare the substrate solution: Prepare TMB substrate by dilutingthree parts of the TMB Substrate solution (K) with 250 parts of QUICKPhosphate-Citrate Buffer (L). It is not necessary to adjust for theslight volume change. For 96 wells (20 ml total volume), add 240 μl ofTMB Substrate Solution (K) to 20 ml of QUICK Phosphate-Citrate Buffer(L) and mix well by swirling in a beaker. Add 40 μl of 3% hydrogenperoxide and mix well.

14. Add TMB substrate solution: Add 200 μl of the TMB substrate solutionprepared in Step 13 to each well. After all of the wells have beenfilled; gently mix by swirling a few times in each direction. Incubateat room temperature for 10 minutes, swirl the plate for 5 seconds, andimmediately read the plate at 650 or 630 nm. If the average OD of thehigh standards is not approximately 0.35, continue the incubation for 2additional minutes and repeat the reading. When the average OD of thehigh standards is between 0.35 and 0.4, add the stop solution.

15. Add the Stop Solution: Add 100 μl of the stop solution (1 M H₂SO₄)to each well. After all of the wells have been filled; gently mix thewell contents by swirling a few times in each direction. Add the stopsolution in the same order and timing as was done for the TMB.

16. Read the plate: Within 30 minutes after adding the stop solution,the plate should be read on a plate reader at 450 nm. For MolecularDevices scanners running SOFTmax software, export the reading as a textfile for data analysis.

***NOTE: This test is time dependent and assumes loading of a platetakes less than 30 minutes at room temperature. If the sample loadingprocedure takes longer than this prescribed time, or if loading multipleplates, pre-plate and transfer samples and standards with amulti-channel pippetor using a new tip for each sample.

Example G Rapid ELISA for PSPB Detection

This antigen-capture, or “sandwich,” ELISA detects PSPB in bovine sera.Serum PSPB binds to antibodies coated in the wells and is detected bysecondary binding of a labeled antibody. Binding of the labeled antibodyconjugate is detected by the addition of the Enhancer and3,3′,5,5′-tetramethylbenzidine (TMB) and is quantified by the subsequentcolor development. A strong color indicates binding and substratereactivity of the labeled antibody conjugate to the bound PSPB, and is apositive indication of pregnancy. Weak color development indicateslittle or no binding of the labeled antibody conjugate due to theabsence of PSPB in the sample, and is a negative indication ofpregnancy. Color development between the standards indicates a recheck.Standards of known concentrations of PSPB are run with each assay andare used to determine the optical density values for assigningpregnant/not pregnant ranges.

Components of the Assay Amounts A Antibody coated plates 5 B SampleBuffer 30 ml C Virgin Heifer Serum (VHS) 4 ml D PSPB High Standard 9 mlE PSPB Low Standard 9 ml F Pregnant Cow Serum (PCS) 4 ml G QUICK Buffer125 ml H B6 rabbit α-bovine PSPB conjugate 750 μl I Enhancer (SA-HRP)400 μl J 20X Wash Buffer concentrate 120 ml K 250X TMB SubstrateSolution 1.5 ml L QUICK Phosphate-Citrate Buffer 125 ml

Other Materials

Single and multichannel adjustable volume pipettors and disposable tips.Multichannel pipettor reservoirs. Microplate reader with the capabilityto read at 650 (or 630) and 450 nm. Deionized or distilled water.Graduated cylinders and beakers. Manual or automatic plate washer.Timer. Platform shaker and shaker/incubator. Microtiter plate covers. 3%hydrogen peroxide. Stop solution (1 M H₂50₄).

Storage and Stability

Store all reagents except for the 20× wash buffer (J) at 4° C. The washbuffer concentrate must be stored at room temperature. Reagents willremain stable when stored as indicated.

Assay Procedure

-   1. Warm up all kit reagents, plates and samples.-   2. Prepare the plates (A). Add 50 μl of Sample Buffer (B) to each    well.-   3. Add controls and samples. Run 150 μl of the controls provided    with the kit as follows. Use a fresh tip for each solution:

Well A-1: VHS (Virgin Heifer Serum) (C) Wells B, C, D-1 PSPB Standard(D) Wells E, F, G-1 PSPB Standard (E) Well H-1: PCS (Pregnant Cow Serum)(F)

-   -   Add 150 μl of serum to the remaining wells. Use a fresh tip for        each sample.

-   4. Incubate the plates. Seal the wells with ParaFilm or DuraSeal,    cover with a microplate lid, and incubate for one hour at 37° C.    with shaking.

-   5. Prepare the Conjugate. Prepare 1× antibody conjugate by diluting    6 parts of the B6 conjugate (H) with 994 parts of QUICK Buffer (G)    (Quick Buffer can be phosphate buffered saline pH 7.2 with 0.5%    bovine serum albumin and 0.05% TWEEN-20 polysorbate 20). For 96    wells, mix 120 μl of the B6 conjugate (H) with 20 ml of QUICK Buffer    (G).

-   6. Prepare the Wash Solution. Prepare 1× Wash Buffer by diluting one    part of the 20× Wash Buffer Concentrate (J) with 19 parts of    deionized or distilled water. Approximately 3 ml/well of 1× wash    solution will be required for this wash step and the wash step below    for a total of 9 washes.

-   7. Wash wells 4 times: After the overnight incubation, remove the    ParaFilm or DuraShield and wash the plate four times. Save the    DuraShield for the next incubation.    -   a. If an automatic washer is used, place the plate on the washer        and wash 4 times with a volume of 300 μl. Set the washer to soak        for 10 seconds and aspirate for 4 seconds between each wash.        Remove any residual wash solution by striking the inverted plate        on a paper towel (blot dry).    -   b. If manual washing is used, dump the contents of the wells        into a sink or tub and then blot dry. Using a multichannel        pipettor or RepeatPipettor, add 300 μl of 1× wash buffer to each        well and swirl the plate for 10 seconds. Repeat the washing        procedure 3 more time (4 washes total).

-   8. Add Conjugate: Using a single- or multi-channel pipettor, add 200    μl of the conjugate from step 5 to each well. Cover with fresh    ParaFilm or the DuraSeal from Step 4, and incubate at 37° C. and 100    RPM for 30 minutes.

-   9. Prepare Enhancer. Prepare 1× Enhancer (SA-HRP) by diluting 2.5    parts of the Enhancer (I) with 997.5 parts of QUICK Buffer (G). For    96 wells, mix 50 μl of the Enhancer (I) with 20 ml of QUICK Buffer    (G).

-   10. Dump, blot and wash wells 1 time. After the 30 minute incubation    listed in Step 8, dump out conjugate solution and blot dry on a    paper towel. Wash plate one time with 1× wash buffer. Dump out the    wash solution and blot dry on a paper towel.

-   11. Add Enhancer. Using a single- or multi-channel pipettor, add 200    μl of the conjugate from step 9 to each well. Cover with fresh    ParaFilm or the DuraSeal from Step 4, and incubate at 37° C. with    shaking for 30 minutes.

-   12. Wash wells 4 times: After the 30 minute incubation listed in    Step 11, repeat the washing procedure described in Step 7, using the    remaining 1× wash buffer (4 washes total).

-   13. Prepare the substrate solution: Prepare 3×TMB substrate by    diluting three parts of the 250×TMB Substrate solution (K) with 250    parts of QUICK Phosphate-Citrate Buffer (L). It is not necessary to    adjust for the slight volume change. For 96 wells (20 ml total    volume), add 240 μl of TMB Substrate Solution (K) to 20 ml of QUICK    Phosphate-Citrate Buffer (L) and mix well by swirling in a beaker.    Add 40 μl of 3% hydrogen peroxide and mix well.

-   14. Add substrate solution: Add 200 μl of the substrate solution    prepared in Step 13 to each well. After all of the wells have been    filled, gently mix by swirling a few times in each direction.    Incubate at room temperature for 15 minutes, swirl the plate for 5    seconds, and immediately read the plate at 650 or 630 nm. If the    average OD of the high standards is not approximately 0.18, continue    the incubation for 2 additional minutes and repeat the reading. When    the average OD of the high standards is between 0.18 and 0.19, add    the stop solution.

-   15. Add the Stop Solution: Add 100 μl of the stop solution (1M    H₂SO₄) to each well. After all of the wells have been filled, gently    mix the well contents by swirling a few times in each direction. Add    the stop solution in the same order and timing as was done for the    TMB.

-   16. Read the plate: Within 30 minutes after adding the stop    solution, the plate should be read on a plate reader at 450 nm. For    Molecular Devices scanners running SOFTmax software, export the    reading as a text file for data analysis.

Note: This test is time dependent and assumes loading of a plate takesless than 30 minutes at room temperature. If the sample loadingprocedure takes longer than this prescribed time, or if loading multipleplates, please pre-plate and transfer samples and standards with amulti-channel pippetor using a new tip for each sample.

Example H Competitive ELISA (cELISA) Protocol Using Progesterone forPregnancy Detection

-   1. Use the NUNC maxisorp plate that has been coated with antibody to    rabbit anti-progesterone antibody and blocked with bovine serum    albumin.-   2. There are Control vials consisting of one vial contains high    progesterone, one vial containing no progesterone.-   3. There are five Standard vials containing 100, 25, 6.25, 1.56, and    0.39 ng/mL progesterone diluted in bull serum.-   4. Prepare conjugate. There is one vial that contains    progesterone-HRP conjugate (at a dilution of 1:100). Prepare a    1:200,000 dilution using the enzyme-linked immunoassay (EIA) buffer    (Phosphate buffered saline, 0.1 M, pH 7.0). For one full plate, add    4.25 μl progesterone-HRP (1:100) to 8.495 mL conjugate buffer. Use    correct lesser proportions if less than a full plate is needed.-   5. Add 30 μl/well of bull serum, each of the five standards of    progesterone, two controls to appropriate wells of the plate.-   6. Add 30 μl/well per well of unknown serum from test animals to    appropriate wells.-   7. Add 70 μl per well of conjugate (prepared as above) using a    multi-channel pipette. Incubate at room temperature for two hours.-   8. Wash the plate 4 times with 300 μl per well each time using wash    buffer (phosphate buffered saline, pH 7.2, 0.02% TWEEN-20    polysorbate 20). Use a multichannel pipette or a plate washer. Wash    buffer is provided as a 10× concentrate. For a full plate, prepare a    dilution of 120 mL by adding 12 mL of 10× to 108 mL of distilled    water.-   9. Prepare 10 mL per full plate of the TMB substrate dilution. Use    the phosphate/citrate buffer (0.05 M Citric Acid, pH 4.0) that is    provided in proportion to the amount of wells that were used in the    plate. Make the dilution by adding 40 μl (4 μl/mL) of sTMB and 20 μl    (2 μl/mL) of hydrogen peroxide (not provided; a 3% solution to be    purchased from a drug store) solution to 10 mL of phosphate/citrate    buffer. Use 100 μL per well.-   10. Incubate for 15 to 20 minutes based upon the color development.-   11. Add 100 μl/well 1M H₂SO₄ to stop the reaction and read at 450    nm.-   12 Calculate the results by using the standard curve after methods    known to those skilled in the art to calculate quantity (ng/mL) of    progesterone in test samples. Apply results known to those skilled    in the art acknowledging the time for sampling the animal after    insemination and quantity of progesterone that is expected in not    pregnant or pregnant animals at time of next expected heat.

Example I Competitive ELISA (cELISA) Protocol for Estrone Sulfate forMares Protocol

1. Use the NUNC maxisorp plate that has been coated with antibody toestrone conjugate and blocked with bovine serum albumin.

2. There are Control vials consisting of one vial contains geldingserum, one vial containing pregnant mare serum and one vial containingnon-pregnant mare serum.

3. There are five Standard vials containing 81, 27, 9, 3, and 1 ng/mLestrone sulfate diluted in gelding serum.

4. Prepare conjugate. There is one vial that contains estronesulfate-HRP conjugate (at a dilution of 1:100). Prepare a 1:350,000dilution using the enzyme-linked immunoassay (EIA) buffer (Phosphatebuffered saline, 0.1 M, pH 7.0). For one full plate, add 2.43 μl estronesulfate-HRP (1:100) to 8.498 mL conjugate buffer. Use correct lesserproportions if less than a full plate is needed.

5. Add 20 μl/well of gelding serum, each of the five standards ofestrone sulfate, pregnant mare serum and open mare serum to appropriatewells of the plate (see plate grid protocol).

6. Add 20 μl/well per well of unknown serum from test animals toappropriate wells.

7. Add 80 μl per well of conjugate using a multi-channel pipette.Incubate at room temperature for two hours.

8. Wash the plate 4 times with 300 ul per well each time using washbuffer (phosphate buffered saline, pH 7.2, 0.02% TWEEN-20 polysorbate20). Use a multichannel pipette or a plate washer. Wash buffer isprovided as a 10× concentrate. For a full plate, prepare a dilution of120 mL by adding 12 mL of 10× to 108 mL of distilled water.

9. Prepare 10 mL per full plate of the TMB substrate dilution. Use thephosphate/citrate buffer (0.05 M Citric Acid, pH 4.0) that is providedin proportion to the amount of wells that were used in the plate. Makethe dilution by adding 40 μl (4 μl/mL) of sTMB and 20 μl (2 μl/mL) ofhydrogen peroxide (not provided; a 3% solution to be purchased from adrug store) solution to 10 mL of phosphate/citrate buffer. Use 100 μlper well.

10. Incubate for 10 to 15 minutes based upon the color development.

11. Add 100 μl/well 1M H₂SO4 to stop the reaction and read at 450 nm.

12. Read the plate at 450 nm on a plate reader. Use the standard curve,after methods known to those skilled in the art to calculate thequantity (ng/mL) of estrone sulfate in test samples.

13. Use the following cutoffs for indication of pregnancy status:

-   -   1. Not pregnant mares or pregnant less than 35 days: 0-5 ng/mL    -   2. Not pregnant or pregnant between 0 and 70 days: >5-13 ng/mL    -   3. Pregnant, questionable value: 9.5-13 ng/mL    -   4. Mares that are 70 or more days in pregnancy: >13 ng/mL

Example J Stabilized Plate Protocol

The stabilizer may improve the shelf-life of the plates that are used inthe assay. Following the below procedure, a plate may maintainimmunological activity for several months rather than 1 to 3 months(with declining immunoactivity) when not stabilized.

For 1 liter of stabilized block solution (25 ml per 96-well plate ifusing 250 uL per well) use the following phosphate buffer containingsalt, sugar and a protein.

0.21 g NaH₂PO4

1.17 g Na₂HPO4

7 g NaCl

pH 7.4

Add 50 grams of sucrose and 5 grams of bovine serum albumin (BSA) perliter.

First, the wells of plates are coated with a BRE (e.g., proteins such asantibodies against PSPB or an antigen such as PSPB). This is done byovernight incubation with the BRE (varied concentrations) in 200microliters (or varied volumes) of bicarbonate buffer, pH 9.4 to 9.6.The plates are then washed four times (or varied times) with wash bufferand are blotted dry. Then 250 ul (or varied volumes but always more thanthe volume of the coating buffer) of stabilized blocker is added to thewells of the plate. Leave the plate on an orbital shaker for one hour.Empty the stabilized blocking solution by inversion and shaking and blotdry. Place the plate in an incubator at 37 degrees centigrade for 2hours for drying. Cover the plate with ParaFilm or a plate sealer asdesired. Alternatively, place un-covered plates into airtight bags (i.e.Mylar foil) that contains a desiccant packet and store at about 4degrees C.

Possible Permutations for a Stabilizer:

Blocking materials: Various proteins (BSA, casein) and/or surfactantTWEEN-20 polysorbate 20, TWEEN-80 polysorbate 80, polyethylene glycol,Triton x-100)

Sugars: Use in combination with non-reducing sugar at 0.25% to 20%(weight/volume). Example of sugars that are non-reducing sugars such assucrose, trehalsoe, etc.,

Buffers: Use of different buffers such as phosphate (as above),carbonate, TRIS, MOPSO, MES.

As for the effectiveness of the stabilizer, after 14 weeks at roomtemperature, a plate containing only dry BSA (a routine method ofblocking) as a blocker had a 50% decrease in ELISA OD signal and a 100%increase in background compared to newly manufactured plates with thedry BSA blocker. The plates stored at room temperature for 14 weeks withthe above “stabilized blocker” had the same signal and background asnewly prepared plates.

Following the above procedure may allow a test surface coated with thestabilizer to be stored at room temperature (about 20° C. (68° F.) toabout 25° C. (77° F.) for about 14 weeks with the test surface havingthe same signal and background as newly prepared test surfaces where thesignal and background were measured within 1 day after being coated.

REFERENCES

All of the following references are specifically incorporated herein byreference:

-   Batistaa A, Beckers J F, Caleroa P, Graciaa A, Gonzalez F,    Sulon J. 2001. Pregnancy-associated glycoproteins (PAG) detection in    milk samples for pregnancy diagnosis in dairy goats. Theriogenology    56:671-676.-   Prakash B, Telugu V L, Walker A M, and Green J A. 2009.    Characterization of the bovine pregnancy-associated glycoprotein    gene family—analysis of gene sequences, regulatory regions within    the promoter and expression of selected genes. BMC Genomics 10:185.    doi:10.1186/1471-2164-10-185-   Butler, J E 1980. Isolation and partial characterization of two    bovine pregnancy-associated proteins. M.S. Thesis. University of    Idaho.-   Butler J E, Hamilton W C, Sasser R G, Ruder C A, Hass G M, Williams    R J: Detection and partial characterization of two bovine    pregnancy-specific proteins. Biol Reprod 26:925-933, 1982.-   Camous S, Sharpigny G, Guillomot M, Martal J, Sasser R G:    Purification of one bovine pregnancy-specific protein by high    performance liquid chromatography (HPLC). Proc. Bard Workshop.    Maternal Recognition of Pregnancy and Maintenance of the Corpus    Luteum, Jerusalem, Abstract 2, 1988.-   Eckblad, W P, Sasser, R G, Ruder C A, Panlasigui P., Kuczynski T.    1985: Localization of pregnancy-specific protein B (PSPB) in bovine    placental cells using glucose oxidase-anti-glucose oxidase    immunohistochemical stain. J. Anim. Sci. 61 (Suppl.): 149-150-   Green J C, Okamura C S, Poock S E and Lucy M C. 2010. Measurement of    interferon-tau (IFN-tau) stimulated gene expression inblood    leukocytes for pregnancy diagnosis within 18-20 d after insemination    in dairy cattle. Anim. Reprod. Sci., 121:24-33.-   Hamilton, W C. 1979. Pregnancy-associated antigens of early    pregnancy in cattle. M. S, Thesis. University of Idaho.-   Hicks B A, Etter S J, Carnahan K G, Joyce M M, Assiri A A, Carling S    J, Kodali K, Johnson G A, Hansen T R, Mirando M A, Woods G L,    Vanderwall D K, and Ott T L. 2003. Expression of the uterine Mx    protein in cyclic and pregnant cows, gilts, and mares. J. Anim. Sci.    81:1552-1561.-   Howard, J M, Gabor G, Gray T,. Passayant C, Ahmadzadeh A, Sasser N,    Pals D, and Sasser S. 2007. BioPRYN a blood based pregnancy test for    managing breeding and pregnancy in cattle. Proc. Western Section    Amer. Soc. Anim. Sci 58:295-297.-   Hughes A L, Green J A, Piontkivska H and Roberts R M. 2003. Aspartic    Proteinase Phylogeny and the Origin of Pregnancy-Associated    Glycoproteins. Molecular Biology and Evolution 20(11):1940-1945.-   Humblot, P., Camous S, Martal J, Charlery J, Jeanguyot N, Thibier M    and Sasser R G. 1988. Diagnosis of pregnancy by radioimmunoassy of a    pregnancy-specific protein in plasma of dairy cows. Thereiogenology    30:257-268.-   Ivani, K A. 1984. Diagnosis of pregnancy by radioimmunoassay of a    pregnancy-specific protein in serum of cows. M.S. Thesis. University    of Idaho.-   King, Cathy. 1996. Presence of pregnancy-specific protein B in milk    of postpartum cows. MS Thesis. University of Idaho.-   Lynch R A, Alexander B M, Sasser R G. 1992. The cloning and    expression of the pregnancy-specific protein B (bPSPB) gene. Biol    Reprod 46(Suppl. 1):72.-   Noyes, J. H., Sasser R G, Johnson B K, Bryant L D, and    Alexander B. 1997. Accuracy of pregnancy detection by serum protein    (PSPB) in elk. Wildlife Society Bulletin 25:695-698.-   Sasser R G, Crock J., Ruder-Montgomery C A. 1989 Characteristics of    pregnancy-specific protein B in Cattle. J Reprod Fertil 37:109-113    (Suppl.).-   Sasser R G, Ruder C A. 1987. Detection of early pregnancy in    domestic ruminants. J Reprod Fertil 34:261-271.-   Sasser R G, Ruder C A, Ivani K A, Butler J E, Hamilton W C. 1986.    Detection of pregnancy by radioimmunoassay of a novel    pregnancy-specific protein in serum of cows and a profile of serum    concentrations during gestation. Biol Reprod 35:936-942.-   Sasser R G, Hamilton W C. U.S. Pat. No. 4,554,256. Novel antigen    associated with early detection of mammalian pregnancy. Nov. 19,    1985.-   Sasser R G, Hamilton W C. U.S. Pat. No. 4,705,748. Antigen    associated with early detection of mammalian pregnancy. A    continuation of U.S. Pat. No. 4,554,256, Nov. 10, 1987.-   Semambo, D K N, Eckersall P D, Sasser R G and Ayliffe T R. 1992.    Pregnancy-specific protein B and progesterone in monitoring    viability of the embryo in early pregnancy in the cow after    experimental infection with Actinomyces pyogenes. Theriogenology    37:741-748.-   Stabenfeldt G H, Daels P F, Munro C J, Kindahl H, Hughes J P,    Lasley B. 1991. An oestrogen conjugate enzyme immunoassay for    monitoring pregnancy in the mare: limitations of the assay between    days 40 and 70 of gestation. J. Reprod. Fertil. 44:37-44.-   Szafranska B., Panasiewicz G, and Majewska M. 2006. Biodiversity of    multiple Pregnancy-Associated Glycoprotein (PAG) family: gene    cloning and chorionic protein purification in domestic and wild    eutherians (Placentalia)—a review. Reprod. Nutr. Dev. 481-502.-   Willard J M, White D R, Wesson C A R, Stellflug J and Sasser    R G. 1995. Detection of fetal twins in sheep using a    radioimmunoassay for pregnancy-specific protein. Journal of Animal    Science 73 960-966.-   Xie S, Low B G, Nagel R J, Kramer K K, Anthony R V, Zoli A P,    Beckers J-F, Roberts R M: Identification of the major    pregnancy-specific antigens of cattle and sheep as inactive members    of the aspartic proteinase family. Proc Natl Acad Sci USA    88:10247-10251, 1991.-   Zoli A P, Beckers J-F, Souters-Ballman P, Closset J, Falmagne P,    Ectors F: Purification and characterization of a bovine    pregnancy-associated glycoprotein. Biol Reprod 45: 1-10, 1991.-   U.S. Pat. Nos. 4,554,256; 4,668,621; 4,705,748; 5,559,097;    6,869,770; 7,393,696; and 7,575,861.-   U.S. Patent Application Numbers 2005/0100975; 2006/0199235;    2007/0166773; 2007/0184558 and 2008/0026384.

Although the present disclosure has been provided with reference to theforegoing operational principles and embodiments, it will be apparent tothose skilled in the art that various changes in form and detail may bemade without departing from the spirit and scope of the disclosure. Thepresent disclosure is intended to embrace all such alternatives,modifications, and variances. Where the disclosure recites “a,” “afirst,” or “another” element, or the equivalent thereof, it should beinterpreted to include one or more such elements, neither requiring norexcluding two or more such elements. Furthermore, any aspect shown ordescribed with reference to a particular embodiment should beinterpreted to be compatible with any other embodiment, alternative,modification, or variance.

What is claimed is:
 1. A method of determining whether a ruminant ispregnant comprising: providing a test kit including at least three testsurfaces, each surface being coated with a pregnancy-specific protein B(PSPB) specific antibody, a first reagent solution including aconjugated PSPB specific antibody, a first standard corresponding to aminimum amount of PSPB present when the ruminant is pregnant, and asecond standard corresponding to a maximum amount of PSPB present whenthe ruminant is not pregnant, obtaining a first blood sample from theruminant after insemination of the ruminant, introducing an amount ofthe first standard to a first one of the test surfaces, introducing anamount of the second standard to a second one of the test surfaces,introducing an amount of the sample to a third one of the test surfaces,allowing the standards and sample to react with the PSPB specificantibody on each test surface under a substantially constant temperaturefor a first specified time period, introducing equal amounts of thefirst reagent solution to each of the three test surfaces, allowing theconjugated PSPB specific antibody to react with the PSPB bound to eachtest surface under a substantially constant temperature for a secondspecified time period, removing any unbound materials from each testsurface, including unbound sample, unbound PSPB, unbound first andsecond standards, unbound first reagent solution, and unbound conjugatedPSPB specific antibody, introducing under a substantially constanttemperature for a specified period of time a visual indicator to undergoa visually detectable change, the magnitude of which is related to theamount of conjugated PSPB specific antibody bound to each surface, andretesting a second blood sample received from the ruminant about 3 daysto about 7 days after the first sample was received if the magnitude ofthe visual indicator on the third surface is between the magnitudes ofthe visual indicators on the first and second surfaces.
 2. The method ofclaim 1, wherein the first standard includes between about 0.1 nanogramsper milliliter to about 1.5 nanograms per milliliter of PSPB in buffer,serum or plasma and the second standard includes between about 0.01ng/ml to about 1.0 nanograms per milliliter of PSPB in buffer, serum orplasma.
 3. The method of claim 1, wherein the first standard includesabout 0.4 nanograms per milliliter of PSPB in buffer, serum or plasmaand the second standard includes about 0.2 nanograms per milliliter ofPSPB in buffer, serum or plasma.
 4. The method of claim 1, furthercomprising between the first and second allowing steps, the step ofremoving any unbound material from each test surface, including unboundsample, unbound PSPB, and unbound first and second standards.
 5. Themethod of claim 1, wherein each test surface is an inner surface of awell that is part of an assay.
 6. The method of claim 1, wherein theruminant is a cow, and the first sample is derived from blood obtainedfrom the cow around 30 days after insemination.
 7. The method of claim6, wherein the cow can be re-inseminated about 1 to about 7 days after anot-pregnant result indication from the first sample or the secondsample.
 8. The method of claim 1, wherein the first and second PSPBspecific antibodies are selected from the group consisting of polyclonalrabbit immunoglobulin, polyclonal goat immunoglobulin, polyclonal sheepimmunoglobulin, polyclonal mouse immunoglobulin, monoclonal mouseimmunoglobulin, and monoclonal rabbit immunoglobulin.
 9. The method ofclaim 1, wherein the PSPB being detected includes one or more variantsof pregnancy associated glycoproteins (PAG).
 10. A test kit fordetecting a selected marker of pregnancy from a sample of an animal,comprising: a first standard solution including a first concentration ofthe selected marker, a second standard solution including a secondconcentration of the selected marker different than the firstconcentration, at least three test surfaces, each test surface coatedwith a biomolecular recognition element selected to bind with theselected marker, a first reagent solution including a conjugatedbiomolecular recognition element selected to bind with the selectedmarker, and a visual indicator selected for its property of producing avisually detectable change under a substantially constant temperatureand a specified reaction time when reacting with the conjugatedbiomolecular recognition element bound to each test surface, wherein thefirst and second concentrations of the marker are selected to generatethe visually detectable change such that a visually detectable changegenerated by the marker from the sample with an intensity greater thanthe first concentration yields a first result, lower than the secondconcentration yields a second result, and between the first and secondconcentrations yields a retest result indicating to retest a secondsample retrieved from the animal at a time period after the firstsample.
 11. The kit of claim 10, wherein the first result is a pregnantresult and the second result is a not pregnant result.
 12. The kit ofclaim 11, wherein a visually detectable change generated by the markerfrom the sample with an intensity equal to the first concentrationyields a pregnant result, and equal to the second concentration yields anot pregnant result.
 13. The kit of claim 10, wherein the marker ispregnancy-specific protein B (PSPB), estrone sulfate, progesterone, oran interferon stimulated gene protein.
 14. The kit of claim 10, whereinthe biomolecular recognition element is a detection antibody and theconjugated biomolecular recognition element is a conjugated detectionantibody.
 15. The kit of claim 14, wherein the kit includes the firstreagent solution including the conjugated detection antibody conjugatedwith a hapten, a second reagent solution with an agent selected to bindwith the hapten-conjugated detection antibody, and a third reagentsolution including the visual indicator selected to bind with the agent.16. The kit of claim 14, wherein the kit includes the first reagentsolution with the conjugated detection antibody conjugated with thevisual indicator.
 17. The kit of claim 14, wherein the kit includes thefirst reagent solution with the conjugated detection antibody conjugatedwith an enzyme, and a second reagent solution including the visualindicator selected to bind with the enzyme-conjugated second detectionantibody.
 18. The kit of claim 10, wherein the biomolecular recognitionelement coated on each well is sealed with a blocking agent, and apreserving agent derived from a stabilizer block solution that includesa non-reducing sugar and a blocking material.
 19. The kit of claim 18,wherein the stabilizer block solution has a pH of about 7.4, thenon-reducing sugar is about 0.25% to about 20% weight to volume, and theblocking material is bovine serum albumin.
 20. The kit of claim 10,wherein the test surfaces are inner surfaces of wells of an array orpart of a test strip.